Acinetobacter O-oligosaccharyltransferases and uses thereof

ABSTRACT

The present application provides methods and uses of O-oligosaccharyltransferase (O-OTases) for generating vaccines. In particular, the present application provides a method of synthesizing a glycoprotein comprising glycosylation of pilin-like protein ComP using a PglLComP O-OTase. Uses of glycoproteins synthesized by glycosylating ComP using PglLComP O-OTase, particularly for the preparation of vaccines and the like, including a vaccine to Streptococcus, is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a 35 U.S.C. § 371 National Phase ofInternational Patent Application No. PCT/CA2016/050208, filed Feb. 26,2016, which claims the benefit of U.S. Provisional Patent ApplicationNo. 62/121,439, filed Feb. 26, 2015, both of which are incorporatedherein by reference in their entireties.

FIELD

The present application pertains to the field of molecular microbiology.More particularly, the present application relates toO-oligosaccharyltransferases, particularly the use of PglL_(ComP)O-oligosaccharyltransferase in vaccine applications.

BACKGROUND

Acinetobacter baumannii and A. nosocomialis are clinically relevantmembers of the Acinetobacter calcoaceticus-A. baumannii (Acb) complexand important opportunistic nosocomial pathogens (Wisplinghoff et al.,2012). These species have emerged as troublesome pathogens due in partto their remarkable resistance to disinfection, desiccation, as well astheir ability to acquire multiply drug resistant phenotypes, all ofwhich promote their survivability in the hospital setting. Furthermore,pan-resistant strains within the Acb are continuously being isolatedfrom hospitals worldwide (Arroyo et al., 2009; Gottig et al., 2014).While the mechanisms of antibiotic resistance of Acb members has beenintensively studied (Gordon et al., 2010), our understanding of theirvirulence mechanisms is unclear. Identified virulence factors include anouter membrane protein A (OmpA), the ability to form biofilms,exopolysaccharide, lipopolysaccharide (LPS), protein glycosylationsystems and capsule (Choi et al., 2008; Choi et al., 2009; Gordon etal., 2010; Iwashkiw et al., 2012; Lees-Miller et al., 2013). A type VIsecretion system (T6SS) has been also identified, although a role inpathogenesis has not been demonstrated (Carruthers et al., 2013; Weberet al., 2013).

A. baylyi is a non-pathogenic member of the genus Acinetobacter,characterized by its genetic tractability and natural competence. Forthese properties, A. baylyi is widely used as a model organism formolecular and genetic studies of the genus Acinetobacter (Vaneechoutteet al., 2006; de Berardinis et al., 2008; Brzoska et al., 2013) and isalso utilized in bioremediation (Abd-El-Haleem et al., 2002; Mara etal., 2012). All members of the Acinetobacter genus, independent of theirpathogenicity, carry a protein glycosylation system (Iwashkiw et al.,2012).

Protein glycosylation, the covalent attachment of carbohydrate moietiesto protein substrates, is the most abundant post-translationalmodification of proteins (Varki, 1993) and occurs in all domains of life(Neuberger, 1938; Sleytr, 1975; Mescher & Strominger, 1976). The majortypes of protein glycosylation are N- and O-glycosylation. Bothprocesses can be classified as oligosaccharyltransferase(OTase)-dependent and OTase independent (Nothaft & Szymanski, 2010;Iwashkiw et al., 2013). OTases are enzymes that catalyze the transfer ofa glycan, previously assembled by cytoplasmic glycosyltransferases (GT)onto an undecaprenyl pyrophosphate lipid carrier, to target proteins.The development of sensitive analytical techniques has led to theidentification of OTase-dependent protein glycosylation in numerousbacterial species. These include members of the genera Campylobacter,Neisseria, Pseudomonas, Francisella, Vibrio, Burkholderia andBacteroides (Szymanski et al., 1999; Faridmoayer et al., 2007;Egge-Jacobsen et al., 2011; Balonova et al., 2012; Gebhart et al., 2012;Coyne et al., 2013; Lithgow et al., 2014). Glycosylation frequentlyaffects protein stability, bacterial adhesion, flagellar filamentassembly, biofilm formation, and virulence in general (Logan, 2006;Iwashkiw et al., 2013). An OTase-dependent, ubiquitous O-linked proteinglycosylation system has been recently discovered within the genusAcinetobacter. This system was required for biofilm formation andpathogenicity of A. baumannii (Iwashkiw et al., 2012). The glycanstructures for several strains of A. baumannii have also beencharacterized and extensive carbohydrate diversity has been established(Scott et al., 2014).

OTases involved in O-glycosylation (O-OTases) do not share extensiveprimary amino acid sequence homologies; yet, all O-Otases containdomains from the Wzy_C superfamily (Power and Jennings, 2003). Orthologsof PglL general 90 O-Otases and WaaL O-antigen ligases are two of themost well characterized enzymes from the Wzy_C superfamily. It hasproven challenging to identify O-OTases based solely on bioinformaticmethodologies as O-OTases and WaaL ligases catalyze similar reactions,i.e. the transfer of lipid-linked glycans to acceptor proteins or lipidA respectively (Hug & Feldman, 2011). The two enzymes appear to beevolutionarily and mechanistically related as mutagenesis oftopologically similar conserved histidine residues of the E. coliO-antigen ligase (H337) and N. meningitidis O-OTase (H349) results inthe loss of glycan transfer activities (Perez et al., 2008; Ruan et al.,2012; Musumeci et al., 2014). Recently, the PglL_A and PglL_B hiddenMarkov models (HMM) were defined to better resolve orthologs of PglLO-OTases from other enzymes of the Wzy_C superfamily (Power et al.,2006; Schulz et al., 2013).

O-OTases are often encoded downstream of their cognate target protein.This genetic arrangement is often found in Gram-negative organismsencoding type IV pili (Tfp) systems, where the major pilin subunit geneis immediately 5′ of the cognate OTase gene (Schulz et al., 2013). Forexample, in P. aeruginosa strain 1244 the major pilin, PilA, isglycosylated by PilO (later renamed TfpO), an O-OTase encodedimmediately downstream of pilA (Castric, 1995; Kus et al., 2004). Thismodification is believed to play a role in virulence asglycosylation-deficient mutants showed decreased twitching motility andwere out-competed by the wild type in a mouse respiratory infectionmodel (Kus et al., 2004; Smedley et al., 2005). The same geneticarrangement and glycosylation phenotype has also been found in P.syringae (Nguyen et al., 2012).

Pilin post-translational modification has also been identified inAcinetobacter species. In A. baylyi ADP1, two Wzy_C superfamilydomain-containing proteins are encoded in the genome. One gene is foundimmediately downstream of the gene encoding the pilin-like protein ComP,whereas the other gene is found within a distant glycan biosynthesisgene cluster. Mutation of the predicted OTase encoded downstream of thecomP gene affected the electrophoretic mobility of ComP, indicating thisgene may encode for a ComP-specific OTase (Porstendorfer et al., 2000;Schulz et al., 2013). Additionally, during the course of a previousstudy demonstrating the functional production of Tfp by the medicallyrelevant A. nosocomialis strain M2, we also identified two molecularforms of PilA differing by apparent molecular weight leading to thehypothesis that the pilins of Acb members may also bepost-translationally modified (Harding et al., 2013; Carruthers et al.,2013).

OTases are powerful tool for glycoengineering conjugate vaccines. Theenzymatic attachment of glycans to proteins present several advantagescompared to the chemical attachment of sugars. Although they exhibitrelaxed specificity, OTases known so far present some limitations. Forexample, glycans containining glucose at the reducing end have not beensuccessfully transferred by any of the known enzymes, such as PglB andPglL. PglB has been shown to required an acetylated sugar at thereducing end (Wacker et al., 2006). PglL was able to transfer sugarswith galactose at the reducing end (Faridmoayer et al., 2008), but ithas not been shown that sugars containing a glucose at the reducing canbe transferred to proteins. This is extremely important for thesynthesis of vaccines against Streptococcus. Most capsular polyccharidesfrom Streptococcus contain a glucose residue at the reducing end(Bentley et al., 2006). The licensed vaccines against S. pneumoniae,such as Prevnar 13, contain up to 13 capsular serotypes. Bettervaccines, containing more serotypes are needed. The current OTases havenot been useful in generating conjugates containing capsule from thesebacteria containing glucose at the reducing end, and therefore they havelittle applications for production of vaccines against Streptococcus.

This background information is provided for the purpose of making knowninformation believed by the applicant to be of possible relevance to thepresent invention. No admission is necessarily intended, nor should beconstrued, that any of the preceding information constitutes prior artagainst the present invention.

SUMMARY

An object of the present invention is to provide the use of an O-OTasefor the generation of glycoproteins for vaccine applications.

In accordance with one aspect of the present invention, there isprovided a method of synthesizing a glycoprotein comprisingglycosylation of ComP using a PglL_(ComP) O-oligosaccharyltransferase.The method can be performed in Gram negative bacteria, such asAcinetobacter (including, for example, A. baylyi, A. baumannii, A.nosocomialis, or A. calcoaceticus) or E. coli, and the like. Theglycosylation can use sugars derived from O-glycosylation, N-glycans, Oantigens, capsular polysaccharides, etc. In one particular embodiment,the N-glycan is derived from Campylobacter, such as Campylobacter jejuniN-hepatasaccharide, for example. Unlike previously known OTases,PglL_(ComP) can be employed to efficiently attach capsularpolysaccharides containing glucose at the reducing end to a proteincarrier. Such capsules are common within the genus Streptococcus. Thepresent application provides the generation of an immune response inmice employing a glycoprotein obtained through the activity ofPglL_(ComP), containing a capsule from S. pneumoniae attached to asuitable carrier. In another embodiment, the ComP can be optionallyfused to a second protein, an adjuvant or a carrier. In another aspectof the present invention there is provided a use of PglL_(ComP) for theglycosylation of a protein, such as a protein with a Streptoccocuscapsule, in particular wherein the Streptococcus is Streptococcuspneumoniae.

In accordance with another aspect of the present invention there isprovided an O-oligosaccharyltransferase (O-OTase) for glycosylation ofComP. The glycosylation can be recombinantly produced in Gram negativebacteria, such as Acinetobacter (including, for example, A. baylyi, A.baumannii, A. nosocomialis, or A. calcoaceticus) or E. coli, and thelike. The glycosylation can use sugars derived from O-glycosylation,N-glycans, O-antigens, capsular polysaccharides, etc. In one particularembodiment, the N-glycan is derived from Campylobacter, such asCampylobacter jejuni N-hepatasaccharide or a capsular polysaccharidefrom Streptococcus, for example. In one particular embodiment, theO-OTase is PglL_(ComP). In another embodiment, the capsularpolysaccharide is a Streptococcus capsule, such as Streptococcuspneumoniae. Thus, the present application provides a vaccine againstStreptococcus, more particularly Streptococcus pneumoniae.

In accordance with a further aspect of the present invention, there isprovided a vaccine comprising a glycoprotein synthesized in accordancewith the method as described herein.

In accordance with yet a further aspect of the present invention, thereis provided a glycoprotein which is a fusion protein comprising ComP ora fragment of ComP carrying a glycosylation site.

BRIEF DESCRIPTION OF THE FIGURES

For a better understanding of the present invention, as well as otheraspects and further features thereof, reference is made to the followingdescription which is to be used in conjunction with the accompanyingdrawings, where:

FIG. 1 illustrates genomic and domain organization of putative O-OTasesof Acinetobacter spp. encoding two OTase genes. (A) Genomic context ofOTases encoded by the A. nosocomialis strain M2 chromosome. (B) Wzy_Csuper family (c104850) and DUF3366 domains present in TfpOM2 and PglLM2.(C) Genomic context of OTases encoded by the A. baylyi ADP1 chromosome.(D) Wzy_C super family (c104850) and DUF3366 domains present inPglL_(ComP) and PglL_(ADP1). The rectangle below PglL_(Comp) indicates aportion of the DUF3366 domain.

FIG. 2 illustrates PilAM2 glycosylation in a TfpO-dependent manner witha tetrasaccharide containing (HexNAc)2, Hexose and N-acetyl-deoxyHexose.(A) Surface proteins from the indicated strains were prepared byshearing, as described in the materials and methods, followed byseparation by SDS-PAGE and western blot analysis of whole cell lysates.PilAM2 from strain M2 was identified employing rabbit anti-PilAM2.PilAM2 from the M2ΔtfpO::kan mutant existed only as a lower molecularform indicating TfpO was required for PilAM2 post-translationalmodification. Strains M2 pilA+ and M2 tfp+ were complemented pilA andtfpO mutants, respectively. “Gly” denotes the glycosylated form of theprotein. (B) PilAM2 was sheared from the surface of strain M2 and ahyper-piliated mutant, precipitated, separated by SDS-PAGE, andvisualized by Coomassie straining. Bands associated with PilAM2 wereexcised and tryptically digested for MS/MS analysis. NSGTDTPVELLPQSFVAS(SEQ ID NO: 91).

FIG. 3 illustrates PilAM2-like glycosylation dependency on a conservedcarboxy-terminal serine. (A) Alignment of the carboxy terminal region ofPilA proteins from P. aeruginosa strain 1244 and selected Acinetobacterstrains (consecutively, SEQ ID NOs: 92-104). All Acinetobacter strainsencoding tfpO homologs contain a carboxy-terminal serine on theirrespective PilA proteins. (B) Western blot analysis of whole cellextracts probing for PilAM2 expression and electrophoretic mobility.Strain M2 derivatives expressing PilA[S132A] and PilA[S136A] wereconstructed and extracts characterized. Serine 132 was not required forglycosylation while serine 136, the C-terminal serine was required forglycosylation. (C) Pilin glycosylation in strain M2 is not required fornatural transformation. Mutants that were unable to glycosylate PilAM2were still naturally transformable.

FIG. 4 illustrates that the major polysaccharide antigen locus (MPA) wasrequired for pilin glycosylation. (A) Genetic organization of the strainM2 MPA locus which is located between the conserved JkpA and lldP genes.Adapted from Hu et al., 2013. (B) Western blot analysis of whole cellextracts probing for PilAM2 expression and electrophoretic mobility fromMPA locus mutants. PilAM2 from the ΔweeH::kan mutant ran at the sameelectrophoretic mobility as PilAM2 from the tfpO::kan mutant indicatingit was not glycosylated. Deletion of the other threeglycosyltransferases yielded PilAM2 proteins with intermediateelectrophoretic mobilities. PilA from the wafY::kan mutant migratedclosest to the WT PilA mobility, then PilA from the wafZ::kan mutant,followed by PilA from the wagB::kan mutant. Mutants that werecomplemented all glycosylated PilAM2.

FIG. 5 illustrates that PglLM2 is a general O-OTase and utilizes thesame lipid-linked glycan donor as TfpOM2. (A) Western blot analysis ofwhole cell extracts probing for OmpA-His expression and electrophoreticmobility. OmpA-His served as bait protein for glycosylation by strain M2as well as the isogenic tfpOM2::kan and pglLM2::kan mutants. All strainsexpressed OmpA-His; however, OmpA-His from the pglLM2::kan mutant ran atan increased electrophoretic mobility indicating the lack ofglycosylation. (B) Glycosylated OmpA-His was purified from solubilizedmembranes using nickel affinity chromatography, separated by SDS-PAGE,and visualized by Coomassie staining. OmpA-His was excised from the geland characterized by MS/MS analysis. AASGVEAAAAPATLTLSTDDK (SEQ ID NO:105).

FIG. 6 illustrates activity of O-OTases in A. baylyi ADP1. (A) Westernblot analysis probing for ComP-His expression in whole cell lysates ofA. baylyi ADP1 as well as the isogenic ΔpglLcmp and ΔpglL_(ADP1)mutants. The increase in ComP-His electrophoretic mobility seen in theΔpglL_(ComP) mutant indicates the absence of pilin glycosylation in thisstrain. (B) Silver stain of LPS obtained from A. baylyi ADP1, theisogenic ΔpglL_(ComP), and ΔpglL_(ADP1) mutants as well as Ralstoniasolanacearum. A. baylyi lacks O-antigen, as seen by the lack ofladdering observed with R. solanacearum LPS. (C, D) Western blotanalysis of whole cell lysates from A. baylyi ADP1, the isogenicΔpglL_(ComP)- and ΔpglL_(ADP1) mutants recombinantly expressinghis-tagged proteins Dsba1 (C) or OmpA-His (D). The increases in therelative mobility of the His-tagged proteins produced in theΔpglL_(ADP1) background indicate that expression of PglL_(ADP1) wasrequired for glycosylation of these proteins.

FIG. 7 illustrates the heterologous expression of TfpO and PglL OTasesin E. coli. Western blot analysis of whole cell lysates of E. coli CLM24expressing, as indicated, the C. jejuni lipid linked oligosaccharide(CjLLO) and His-tagged DsbA1 together with an A. baylyi or A. baumanniiATCC 19606 OTase. His-tagged DsbA1 was detected using the polyclonalanti-his antibody (green) and CjLLO was detected using the hR6 antibody(red). Co-localization of both signals, seen in yellow, indicatesglycosylation of DsbA1 by the Campylobacter oligosaccharide. This isseen only when PglL_(ADP1) or PglL₁₉₆₀₆ were expressed in E. coli CLM24along with CjLLO and His-tagged DsbA1.

FIG. 8 provides an O-glycan structure identified using ZIC-HILICenrichment of A. baylyi ADP1 glycoproteins. ITMS-CID fragmentationresults in near exclusive glycan fragmentation of A. baylyi ADP1glycopeptides enabling the identification of four unique glycans onmultiple protein substrates corresponding to; A and D) a pentasaccharidecomposed of 286-217-HexNAc3 (1112.41 Da, 92DAAHDAAASVEK 103 (SEO ID NO:106) of Q6FCV1_ACIAD); B, F and G) two isobaric glycoforms composed of286-217-245-HexNAc2 and 286-217-HexNAc-245-HexNAc (1154.41 Da,344NTAASSVAATHKK356 (SEQ ID NO: 107) of Q6F814_ACIAD) and C and E) apentasaccharide composed of 286-217-2452-HexNAc (1196.41 Da,344NTAASSVAATHKK356 (SEQ ID NO: 107) of Q6F814_ACIAD).

FIG. 9 illustrates that PglL_(ComP), but not TfpOM2, is specific for itscognate pilin protein. (A) Western blot analysis of whole cell extractsprobing for heterologous PilAM2 expression and electrophoretic mobility.PilAM2 was glycosylated in A. baumannii ATCC 19606 and A. baumannii27413, both of which encode tfpO homologs. Strains lacking tfpO homologs(A. baumannii ATCC 17978 and A. baylyi ADP1) were unable to glycosylatePilAM2. (B) Western blot analysis probing for heterologous ComP-Hisexpression and electrophoretic mobility. ComP-His was only modified inA. baylyi ADP1 indicating that PglL_(ComP) is specific for ComP. (C)Western blot analysis probing for heterologous PilA17978 expression andelectrophoretic mobility. PilA17978 was glycosylated in its nativestrain by PglL17978 and in A. baylyi ADP1 by PglL_(ADP1), but not byPglL_(ComP).

FIG. 10 illustrates a model of lipid-linked oligosaccharide synthesis,TfpOM2-dependent pilin glycosylation, and PglLM2 general O-glycosylationin A. nosocomialis strain M2. The proteins encoded by the genes from themajor polysaccharide antigen locus synthesize the tetrasaccharide(HexNAc)-(Hex)-(deoxy-Hex)-(HexNAc) on an undecaprenyl lipid carrier,which is then transferred to the periplasm. The lipid-linkedoligosaccharide can then be transferred to the major pilin protein,PilA, by the pilin-specific OTase TfpO or further processed andtransferred to other proteins, such as, OmpA by the general OTasePglLM2.

FIG. 11 illustrates the structures of the glycans employed in thepresent application. Structures are modified from Faridmoayer et al.,2008 and van Selm et al., 2003.

FIG. 12 illustrates PglL_(ComP) transfers C. jejuni LLO and E. coli O7antigen to ComP. Western blot analyses on E. coli CLM24 whole celllysates expressing ComP with or without PglL_(ComP) coexpressed with (A)C. jejuni LLO and (B) E. coli O7 antigen. Lane 1 corresponds tounglycosylated ComP, with no OTase coexpressed. In lane 2, ComP andPglL_(ComP) are coexpressed. (A) Lower electrophoretic mobility bandsthat react to the anti-his and anti-glycan antibodies indicateglycosylation of ComP by the C. jejuni LLO. (B) Lower electrophoreticmobility bands that react to the anti-his indicate glycosylation by E.coli O7 antigen subunits. Glycoprotein signals disappear upon ProteinaseK digests (lane 3). Expression was probed for with a monoclonal anti-hisantibody.

FIG. 13 is an illustration that PglL_(ComP), but not NmPglL or CjPglB,can transfer CPS subunits from S. pneumoniae serotype 14 to ComP.Western blot analyses of (Lane 1) whole cell lysates of E. coli CLM24expressing his-tagged (A) ComP, (B) DsbA and (C) AcrA. Lane 2corresponds to Ni-NTA purified proteins from E. coli CLM24 coexpressingthe S. pneumoniae serotype 14 CPS synthesis locus and PglL_(ComP). Lowerelectrophoretic mobility bands in panel A that react to both Anti-Hisand Anti-Glycan antibodies in lane 2 relative to lane 1 indicateglycosylation of ComP by CPS subunits in a PglL_(ComP)-dependent manner.Lane 2 in panels B and C shows no lower electrophoretic mobility bandscompared to lane 1, indicating the inability of NmPglL and CjPglB totransfer CPS subunits to DsbA1 and AcrA respectively. Expression wasprobed for with a monoclonal anti-his antibody and a polyclonalanti-glycan antibody.

FIG. 14 is an illustration of visual results of whole cell ELISAsperformed on post immune mouse sera obtained after 21 days. ELISAs weredone using sera from (A) mice immunized with the unglycosylated proteinand probed for with a secondary HRP-conjugated anti-IgM antibody, (B)mice immunized with the glycosylated protein and probed for with asecondary HRP-conjugated anti-IgM antibody, or (C) mice immunized withthe unglycosylated protein and probed for with a secondaryHRP-conjugated anti-IgG antibody.

FIG. 15 is a graphical illustration of sera from mice, when injectedwith CPS-conjugated ComP, react against S. pneumoniae whole cells. Mousesera from 10 animals (pre immune bleeds and post immune day 21) wereincubated in wells of 96 well plates containing immobilized heat-killedwhole cell S. pneumoniae serotype 14. As a negative control, sera from10 mice injected with unglycosylated protein were tested. SecondaryHRP-conjugated antibodies used were against mouse IgG antibodies formice injected with the unglycosylated protein, or against mouse IgM andmouse IgG antibodies for mice injected with the CPS-conjugated ComP.This was followed by treatment with the chromogenic substrate TMB. Serafrom mice probed for with the HRP-conjugated mouse IgG antibody showedan increase in absorbance values at 650 nm compared to mice injectedwith the unglycosylated protein.

FIG. 16 provides a graphical representation of immune responses. Panel(A) illustrates the combined data from whole cell ELISAs against mousesera. Panel (B) illustrates absorbance values at 650 nm of TMB-treatednegative control (no primary antibody) or positive control wells(commercially available rabbit antibody against the S. pneumoniaeserotype 14 capsular polysaccharide).

FIG. 17 demonstrates that the IgG immune response observed is directedagainst CPS from S. pneumoniae serotype 14. Western blot analyses of LPSobtained from E. coli CLM37 probing for expression of S. pneumoniaeserotype 14 CPS with the mouse sera followed by a secondary fluorescentmouse anti IgG antibody. (A) Sera from the negative control miceinjected with the unglycosylated protein did not react to CPS. (B) Seraobtained from a mouse immunized with the CPS-conjugated ComP thatreacted in the ELISA plates reacted to CPS in the Western blots. Acommercial anti-CPS rabbit antibody was used as a positive control.

FIG. 18 provides a summary of the current knowledge of the glycanstransferred by CjPglB and NmPglL to their acceptor proteins incomparison with PglL_(ComP).

FIG. 19 illustrates quantitative analysis of glycosylation in A. baylyiADP1 WT, A. baylyi ADP1ΔpglL_(ADP1), and A. baylyi ADP1ΔpglL_(comP)using dimethyl labeling. Using dimethyl labeling and ZIC-HILIC, theO-OTase responsible for glycosylation of individual glycopeptides wasconfirmed. Glycopeptides derived from A. baylyi ADP1 WT, labeled withlight label and A. baylyi ADP1ΔpglL_(comP) labeled with heavy label,were observed at near 1:1 levels; whereas, A. baylyi ADP1ΔpglL_(ADP1),labeled with medium label, was undetectable within samples. Converselynon-glycosylated peptides were observed at a near 1:1:1 level betweenall three strains. A and D) The MS spectra of the light, medium andheavy isotopologues of the glycopeptide 113KLAEPAASAVADQNSPLSAQQQLEQK138(SEQ ID NO: 108) (Q6F825_ACIAD) and non-glycosylated peptide166AQSVANYLSGQGVSSR182 (SEQ ID NO: 109) (Q6FDR2_ACIAD) enabled thecomparison of glycosylation across all three strains. No glycopeptideswere observed within ADP1ΔpglL_(ADP1) while non-glycosylated peptideswere observed a near 1:1:1 ratio. B and E) Comparison of the extractedion chromatograms of the light, medium and heavy isotopologues confirmthe absent of ADP1ΔpglL_(ADP1) derived glycopeptides and the 1:1:1 ratioof non-glycosylated peptides. C) HCD fragmentation confirming theidentification of the heavy isotopologues of the glycopeptide113KLAEPAASAVADQNSPLSAQQQLEQK138 (SEQ ID NO: 108), confirming itsorigins from ADP1ΔpglL_(comP). F) HCD fragmentation confirming theidentification of the medium isotopologues of the non-glycosylatedpeptide 166AQSVANYLSGQGVSSSR182 (SEQ ID NO: 109), confirming its originsfrom ADP1ΔpglL_(ADP1).

FIG. 20 illustrates genomic organization of OTase(s) in (A) A. baumanniiATCC 17978 and (B) A. baumannii ATCC 19606.

FIG. 21 illustrates quantitative glycopeptides identified in A. baylyiADP1. (SEQ ID NOs: 79-82).

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise.

The term “comprising” as used herein will be understood to mean that thelist following is non-exhaustive and may or may not include any otheradditional suitable items, for example one or more further feature(s),component(s) and/or ingredient(s) as appropriate.

A genomic analysis of sequenced genomes of Acinetobacter spp. revealedthat, in addition to A. baylyi ADP1, multiple strains within the genusAcinetobacter encode two OTases. In accordance with the presentapplication there are provided genetic and proteomic techniques todemonstrate that both OTases are functional and that one of theseenzymes acted as a pilin-specific OTase, whereas the other OTase wasable to glycosylate a wide range of proteins. In addition, using massspectrometry, the glycan structure of A. nosocomialis strain M2 wascharacterized and the glycoproteome of A. baylyi defined.

In recent years, a panoply of glycosylation pathways have beenidentified in bacteria. Irrespective of the pathway utilized, both N-and O-glycans often decorate cell surface adhesins in both Gram-negativeand Gram-positive bacteria. Examples of glycosylated surface-associatedproteins include the O-glycosylation of AIDA-I and TibA in E. coli(Charbonneau et al., 2007; Cote et al., 2013), the type IV pilins ofPseudomonas, Neisseria, Dichelobacter nodosus, and Fransicellatularensis (Castric et al., 1995, Aas et al., 2006; Faridmoayer et al.,2007, Voisin et al. 2007, Cagatay et al., 2008, Egge-Jacobsen et al.,2011), the flagellins in multiple bacterial species (Nothaft &Szymanski, 2010; Iwashkiw et al., 2013), the serine rich adhesins inStreptococcus spp. (Zhou & Wu, 2009), and the N-glycosylation of HMWG inHaemophilus (Gross et al., 2008) and Aggregatibacteractinomycetemcomitans (Tang & Mintz, 2010). Medically relevant Acbmembers are not the exception and the present application, as well asprevious work, show that pilin and multiple outer membrane proteins areO-glycosylated, which could provide an adherence advantage either tohost cells, in bacterial communities, or to abiotic surfaces. However,as of yet, the biological role for glycosylation of Acinetobacter pilinsubunits has not been elucidated. Specifically, the A. nosocomialisstrain M2ΔtfpO::kan mutant was equally as transformable as the parentalstrain (FIG. 3C), exhibited the same twitching motility phenotype as theparent strain, and also contained similar levels of surface 428 exposedPilA (data not shown). Furthermore, no condition was found in which A.baylyi ADP1 would attach to abiotic surfaces or form biofilms.

As mentioned previously, pilin glycosylation in P. aeruginosa and P.syringae, mediated by PilO and TfpO, respectively, was essential formotility, biofilm formation and virulence (Smedley et al., 2005; Nguyenet al., 2012). Pilin glycosylation had no effect on natural competencein A. baylyi (Porstendorfer et al., 2000) or A. nosocomialis (FIG. 3C).Nevertheless, the ubiquitous nature of O-linked protein glycosylationwithin the genus Acinetobacter suggests a key, still unknown role forthis post-translational modification.

As described herein, some Acinetobacter strains encode two OTasehomologs, one of which is required for general O-glycosylation and theother that specifically modifies pilin. The majority of the medicallyrelevant Acinetobacter strains, including A. nosocomialis strain M2,encode two contiguous OTases, which are located immediately downstreamof a type IVa major pilin subunit gene. At the time of the presentapplication, 76% of A. baumannii isolates with completed genomes encodeda PilA protein containing a carboxy-terminal serine. All isolatescontaining a gene encoding a PilA protein with a carboxy-terminal serinealso encode for a tfpO homolog found immediately downstream of pilA.This finding is congruous with findings reported for the group I pilins(PilAI) found in P. aeruginosa (Kus et al., 2004). Thus there appear tobe multiple lineages of pilin genes, specifically, a lineage thatcontains an allele that encodes for a PilA with a carboxy-terminalserine and the downstream accessory gene tfpO and lineages that are notglycosylated by a TfpO-like activity. All isolates lacking acarboxy-terminal serine on the major pilin protein, including ATCC17978, do not encode for a tfpO homolog consistent with the separateevolution of Acinetobacter pilin lineages.

In contrast to the contiguous organization of OTases in the medicallyrelevant Acinetobacter spp., the two OTases of the environmental isolateA. baylyi ADP1 are distantly separated on the chromosome. Schulz et al.(2013) showed that the OTase homolog pglL_(ComP), which is encodedadjacent to comP, is responsible for ComP modification. Mutationalanalysis coupled with an in vivo glycosylation assay as well as thecharacterization of the glycoproteome demonstrated that pglL_(ComP) is aComP-specific OTase. On the other hand, the second OTase, PglLADP1, isnot an O-antigen ligase as previously suggested but rather a generalO-OTase glycosylating multiple protein targets.

The present application provides that PglLM2, encoded by M215_10475, isable to recognize the same motif that the general O-OTase PglL found inall other A. baumannii strains recognizes, as evidenced by the abilityof A. nosocomialis M2 to glycosylate OmpA-His. In A. nosocomialis strainM2, both PglLM2 and TfpOM2 utilize the same lipid linked tetrasaccharideto modify their target proteins. The present application provides thatPglLM2 was able to transfer two subunits of the glycan, whereas TfpOM2only transferred a single glycan chain. Previous studies have shown twosubunits of the glycans being transferred by general OTases (Scott etal., 2014); and that TfpO is unable to transfer long sugar chains to P.aeruginosa pilin (Faridmoayer et al., 2007). Furthermore, the MPA locuswas identified as the source of the genes encoding the proteinsresponsible for the synthesis of the shared lipid-linkedtetrasaccharide. FIG. 10 provides an exemplary model depicting O-glycansynthesis by the MPA cluster and the shared usage of this lipid-linkedglycan by TfpOM2 and PglLM2.

Although many protein glycosylation systems have been identified, howO-OTases, such as the ones from A. baumannii, Neisseria spp. andBurkholderia spp., recognize the acceptor sequences in their proteintargets is still not clear. It has been established that OTasesrecognize low complexity regions (LCR), rich in serine, alanine andproline (Vik et al., 2009). The pilin specific TfpO enzymes describedhere recognize a peptide of about 15 amino acids containing many serineand proline residues. Similarly to P. aeruginosa TfpO, it may besuggested, in accordance with the present application, that thecarboxy-terminal serine of PilAM2 may serve as the site ofTfpOM2-dependent glycosylation. Bacterial species carrying twofunctional O-OTases, a PglL-general OTase and a pilin-specific OTasehave not previously been identified. TfpO is the only OTase present inPseudomonas (Smedley et al., 2005; Nguyen et al., 2012), while PglL isthe only OTase identified in Neisseria (Faridmoayer et al., 2007), A.baumannii ATCC 17978 (Iwashkiw et al., 2012), B. cenocepacia K56-2(Lithgow et al., 2014) and R. solanacearum (Elhenawy et al., submitted).Three possible O-OTases have been identified in V. cholerae, but theactivity of only one of these has been shown in E. coli (Gebhart et al.,2012), and no glycoproteins have been identified in V. cholerae. In N.meningitidis and N. gonorrhoeae, the OTase PglL is able to glycosylatepilin and several other proteins (Aas et al., 2006; Faridmoayer et al.,2007). Although PglL can recognize three glycosylation sites in pilinwhen the system is reconstituted in E. coli, none of them contain thetypical LCR domain found in the remaining Neisseria glycoproteins,indicating that PglL can recognize more than one motif (Musumeci et al.,2014).

In Francisella spp. the OTase is closely related to PilO/TfpO and itappears to be responsible for both pilin and general glycosylation(Balonova et al., 2012). Why Acinetobacter strains require two differentOTases to glycosylate pilin and other proteins remains unclear as somepathogenic strains of A. baumannii carry only PglL, which is requiredfor optimal biofilm formation and virulence (Iwashkiw et al., 2012). Itis important to note that non-pathogenic A. baylyi ADP1 also containstwo O-OTases. However there are several differences between theComP-specific OTase PglL_(ComP) of A. baylyi and the pilin-specificOTases TfpO of the medically relevant Acinetobacter spp. Although bothOTases are encoded immediately downstream of their cognate proteinacceptors (FIG. 1 panels A and C), TfpO OTases are hypothesized to bespecific for the carboxy-terminal serine present on PilA, as acarboxy-terminal serine to alanine point mutant was unable to produceglycosylated pilin. Interestingly, all Acinetobacter strains encoding atfpO gene homolog also contained the carboxy-terminal serine on theirrespective PilA sequences. Furthermore, the present applicationdemonstrates that Acinetobacter TfpO homologs are functionallyexchangeable as PilAM2 was modified by each tfpO encoding strain tested(FIG. 9A). The variable electrophoretic mobility of PilAM2 is likely dueto glycan variability between these strains (Scott, et al., 2014).

Although the site of ComP glycosylation has not been identified, it ispredicted to be at an internal residue as ComP does not contain acarboxy terminal serine or any carboxy terminal residue associated withpost-translational modification. BLAST analysis of the ComP-specificOTase also demonstrated that PglL_(ComP) is more closely related to thegeneral OTase PglLM2 than to TfpO. Although the pilin-specific TfpOOTase could cross glycosylate different pilins containingcarboxy-terminal serines, PglL_(ComP) was unable to glycosylate thepilins recognized by TfpO.

In accordance with the present application, three different classes ofOTases are found to be present in Acinetobacter: the pilin-specific TfpOenzymes that glycosylate pilins containing carboxy-terminal serineresidues; the general PglL OTases that recognize LCR in multipleproteins; and PglL_(ComP), which appears to be exclusively devoted tothe glycosylation of ComP. These enzymes have different biochemicalcharacteristics, which provide helpful information for the synthesis ofnovel glycoconjugates with biotechnological applications. Thedifferentiation between these enzymes is not trivial, and may not beaccurately predicted just by the presence of pfam domains. For example,despite having the highest degree of sequence similarity and beingfunctionally homologous, PilO/TfpO from P. aeruginosa strain 1244contains the pfam04932 domain, whereas tfpO from A. nosocomialis strainM2 contains the pfam13425 domain. Moreover, the general PglL OTases ofthe medically relevant Acinetobacter spp., including strain M2 and A.baumannii ATCC 17978, contain domains from the pfam04932 family and theA. baylyi general PglL_(ADP1) OTases contain a pfam13425 domain. Addingto the complexity is the fact that the general PglL OTases frommedically relevant Acinetobacter spp. and the A. baylyi ComP-specificPglL_(ComP) contain the same pfam04932 domains yet recognize differentsequons. In addition, this pfam domain is present in the WaaL O-antigenligases. While bioinformatic analyses can be powerful tools to initiallylocate and identify ORFs encoding proteins predicted to be involved inglycan transfer events, the present application reinforces the conceptthat the activity of bioinformatically identified O-OTases must beexperimentally determined and reveals a complex and fascinatingevolutionary pathway for bacterial O-OTases.

In accordance with the present application, and unlike previously knownOTases, PglL_(ComP) can be employed to efficiently attach capsularpolysaccharides containing a glucose residue at the reducing end, to acarrier protein, such as ComP. Such capsules are common within the genusStreptococcus. Expression of PglL_(ComP) in E. coli in presence ofplasmids expressing ComP and a capsular polysaccharide from S.pneumoniae resulted in the glycosylation of ComP with saidpolysaccharide. Injection of this glycoprotein in mice mounted aspecific IgG immune response against the capsular polysaccharide,demonstrating the applicability of PglL_(ComP) to generate recombinantconjugate vaccines against Streptococcus.

To gain a better understanding of the invention described herein, thefollowing examples are set forth. It should be understood that theseexamples are for illustrative purposes only. Therefore, they should notlimit the scope of this invention in any way.

EXAMPLE 1

Materials and Methods

Strains, Plasmids, and Growth Conditions.

A list of bacterial strains and plasmids used herein are as follows:

TABLE 1 Plasmids and bacterial strains used herein Reference/ Plasmid orstrain Relevant characteristic(s) Source PLASMIDS pFLP2 Encodes FLPrecombinase Kumar et al., (2010) pKD13 Contains kanamycin resistancegene from Tn5 flanked by Datsenko & FRT sites Wanner, (2000) pRSM3542pKD13 containing kan-sacB Carruthers et al., (2013) pGEM-T-Ez Generalcloning plasmid Promega pCC1 Single copy, general cloning plasmidEpicentre pSMART-LCKAN Low copy blunt cloning vector Lucigen pGEM-pilApGEM containing pilA with 1 kb flanking DNA Harding et al. (2013)pGEM-pilA::kan-sacB pGEM-pilA containing pilA::kan This studypCC1-pilA-tfpO-pglL pCC1 containing the pilA-tfpO-pglL locus with Thisstudy approximately 1 kb of flanking DNA pRSM3510 pKNOCK derivative witha mini-Tn7 element containing a Harding et al. multiple cloning site(2013) pRSM3510-pilA pRSM3510 containing pilA with expression drivenfrom the Harding et al. predicted pilA promoter (2013)pRSM3510-pilA[S136A] pRSM3510-pilA with a carboxy terminal serine toalanine This study point mutation pRSM3510-pilA[S132A] pRSM3510-pilAwith a serine 132 to alanine point mutation This studypRSM3510-pilA^(P)-tfpO pRSM3510 containing the predicted pilA promoter,the ATG This study of pilA, a FLP scar, the last 21 bp of pilA, and thetfpO gene including the 48 bp intergenic region between pilA and tfpOpCC1-pglL::kan pCC1-pilA-tfpO-pglL containing pglL::kan This studypGEM-weeH::kan pGEM-T-Ez containing weeH::kan This study pRSM4063pSMART-LCKan containing an the empty mini-Tn7 element This study frompRSM3510 along with 2 kb of flanking DNA up and downstream of the attTn7from strain M2 pRSM4063-weeH pRSM4063 containing the weeH gene with itspredicted This study promoter pWH1266 E. coli - Acinetobacter shuttlevector Hunger et al., (1990) pGEM-wafY::kan pGEM-T-Ez containingwafY::kan This study pGEM-wafZ::kan pGEM-T-Ez containing wafZ::kan Thisstudy pGEM-wagB::kan pGEM-T-Ez containing wagB::kan This study pCC1-GTpCC1 containing the predicted promoter of the wxy gene This study (329bp upstream), wxy, wafY, wafZ, wagA, gnaB, and wagB pCC1-wxy^(P)-wafYpCC1-GT lacking the wxy open reading frame This study pCC1-wxy^(P)-wafZpCC1-GT lacking the wxy and wafY open reading frames This studypCC1-wxy^(P)-wagB pCC1-GT lacking the wxy, wafY, wagA, and gnaB openThis study reading frames pRSM4063-wxy^(P)-wafY pRSM4063 containing wafYdriven off the predicted wxy This study promoter pRSM4063-wxy^(P)-wafZpRSM4063 containing wafZ driven off the predicted wxy This studypromoter pRSM4063-wxy^(P)-wagB pRSM4063 containing wagB driven off thepredicted wxy This study promoter pWH-pilA_(M2) pWH1266 expressingpilA_(M2) driven by the predicted pilA This study promoterpRSM3510-A1S_1193-his pRSM3510 containing A1S_1193 driven off itspredicted This study native promoter pET-15b General plasmid forexpression and cloning of recombinant Novagen proteins based on theT7-promoter driven system pET-15b-rsPilA_(M2) pET-15b expressing atruncated pilA from the T7 promoter This Study pEXT20 Amp^(r) cloningand expression vector, IPTG inducible. Dykxhoorn et al., (1996) pBAVMCSKm^(r) pBAV1K-T5-gfp derivative with gfp ORE removed. Nakar &Constitutive E. coli/Acinetobacter shuttle vector Gutnick, (2001)pBAV-comP-his Km^(r) pBAVmcs constitutively expressing C-6X His-taggedThis Study comP from A. baylyi, inserted at BamHI and SalI.pWH-pilA₁₇₉₇₈-His pWH1266 expressing pilA₁₇₉₇₈ driven by the predictedpilA This study promoter pEXT-pglL_(comP) Amp^(r) pEXT20 expressing C-6XHis-tagged pglL_(ComP) from This Study A. baylyi inserted at BamHI andSalI, IPTG inducible. pEXT-pglL_(ADP1) Amp^(r) pEXT20 expressing C-6XHis-tagged pglL_(ADP1) from This Study A. baylyi inserted at BamHI andSalI, IPTG inducible. pEXT-tfpO₁₉₆₀₆ Amp^(r) pEXT20 expressing C-10XHis-tagged tfpO₁₉₆₀₆ from This Study A. baumannii ATCC 19606 inserted atBamHI and SalI, IPTG inducible. pEXT-pglL₁₉₆₀₆ Amp^(r) pEXT20 expressingC-10X His-tagged pglL₁₉₆₀₆ from This Study A. baumannii ATCC 19606inserted at BamHI and SalI, IPTG inducible. pAMF22 Tp^(r) C-10XHis-tagged dsbA1 from N. meningitidis MC58 Faridmoayer cloned intopMLBAD, Arabinose inducible. A. and Feldman M F. (unpublished)pBAV-dsbA1-His C-6X His-tagged dsbA1 subcloned into pBAVMCS, Km^(r), atThis Study BamHI and HindIII. Constitutively expressing. pACYCpglBCm^(r) pACYC184-based plasmid encoding the C. jejuni Wacker et al.,protein glycosylation locus cluster with mutations W458A (2002) andD459A in PglB, IPTG inducible. pBAVMCS- Km^(r) pBAVMCS constitutivelyexpressing C-10X hist- Scott et al., A1S_1193His10X tagged A1S_1193inserted at BamHI and SalI. (2014) STRAINS Acinetobacter nosocomialisMetro Health Systems Clinical Isolate Niu et al., strain M2 (2008)M2ΔpilA::kan Strain M2 containing a deletion of pilA and replacementwith Harding et al. a kanamycin resistance cassette (2013)M2ΔpilA::kan-sacB Strain M2 containing a deletion of pilA andreplacement with This study a kan-sacB cassette M2ΔpilA Strain M2containing an unmarked, in-frame deletion of pilA This study M2ΔpilTStrain M2 containing an unmarked, in-frame deletion of pilA Harding etal. (2013) M2ΔtfpO::kan Strain M2 containing a deletion of tfpO andreplacement This study with a kanamycin resistance cassetteM2ΔtfpO::kanΔpilT::strep M2ΔtfpO::kan containing a deletion of pilT andreplacement This study with a streptomycin resistance cassetteM2ΔpglL::kan Strain M2 containing a deletion of pglL and replacementThis study with a kanamycin cassette M2ΔpilA (pilA[S136A]+) M2ΔpilA::kanwith a mini-Tn7 element containing an allele This study of pilA with acarboxy-terminal serine to alanine point mutation M2ΔwafY::kan Strain M2containing a deletion of wafY and replacement This study with akanamycin resistance cassette M2ΔwafZ::kan Strain M2 containing adeletion of wafZ and replacement This study with a kanamycin resistancecassette M2ΔwagB::kan Strain M2 containing a deletion of wagB andreplacement This study with a kanamycin resistance cassette M2ΔweeH::kanStrain M2 containing a deletion of weeH and replacement This study witha kanamycin resistance cassette M2ΔpilA::kan (pilA+) M2ΔpilA::kan with amini-Tn7 element containing the pilA Harding et al. gene transcribedfrom its predicted promoter (2013) M2ΔtfpO::kan (tfpO+) M2ΔtfpO::kanwith a mini-Tn7 element containing the tfpO This study gene transcribedfrom the pilA predicted promoter M2ΔwafY::kan (wafY+) M2ΔwafY::kan witha mini-Tn7 element containing the wafY This study gene under control ofthe predicted wxy promoter M2ΔwafZ::kan (wafZ+) M2ΔwafZ::kan with amini-Tn7 element containing the wafY This study gene under control ofthe predicted wxy promoter M2ΔwagB::kan (wagB+) M2ΔwagB::kan with amini-Tn7 element containing the This study wafY gene under control ofthe predicted wxy promoter M2ΔweeH::kan (weeH+) M2ΔpilA::kan with amini-Tn7 element containing the pilA This study gene fused to a FLAG tagtranscribed from its predicted promoter M2 (A1S_1193-his+) Strain M2with a mini-Tn7 element containing A1S_1193- This study his transcribedfrom its predicted promoter M2ΔtfpO::kan (A1S_1193- M2ΔtfpO::kan with amini-Tn7 element containing This study his+) A1S_1193-his transcribedfrom its predicted promoter M2ΔpglL::kan (A1S_1193- M2ΔpglL::kan with amini-Tn7 element containing This study his+) A1S_1193-his transcribedfrom its predicted promoter A. baumannii ATCC 17978 Reference A.baumannii strain ATCC A. baumannii ATCC 19606 Reference A. baumanniistrain ATCC A. baumannii 27413 A. baumannii clinical isolate isolated atNationwide NCH Children's Hospital (NCH) from body fluid A. baylyi ADP1Environmental isolate de Berardinis et al., (2008) A. baylyiΔpglL_(ComP)::kan Strain ADP1 with pglL_(comP) deleted and replaced witha de Berardinis kanamycin resistance casette et al. (2008) A. baylyiΔpglL_(ADP1)::kan Strain ADP1 with pglL_(ADP1) deleted and replaced witha de Berardinis kanamycin resistance casette et al. (2008) E. coli DH5aGeneral cloning strain Invitrogen E. coli EC100D pir+ General cloningstrain, pir⁺ Epicentre E. coli DY380 Recombineering strain Lee et al.,(2001) E. coli DH5a(pFLP2) Carries FLP recombinase gene undertemperature control Kumar et al. (2010) E. coli HB101(pRK2013)Conjugation helper strain Figurski & Helinski, (1979) E. coliEC100D(pTNS2) Carries transposase genes for mini-Tn7 transposition Choiet al., (2005) E. coli Origami 2(DE3) K-12 derivative containingmutations in trxB and gor genes Novagen and a host lysogen of λDE3 E.coli Stellar chemically HST08 strain derivative for high transformationefficiencies Clontech competent cells E. coli CLM24 Constructed from E.coli W3110 (IN(rrnD-rrnE)1 rph-1). Feldman et waaL mutant al., (2005)

All bacteria were grown on L-agar or in LB-broth at 37° C. unlessotherwise noted. When appropriate, antibiotics were added to the A.nosocomialis or A. baumannii cultures at the following concentrationsexcept when noted otherwise: 100 μg ampicillin/mL, 20 μg kanamycin/mL,or 12.5 μg chloramphenicol/mL. When appropriate, E. coli cultures weresupplemented with antibiotics at the following concentrations: 50 μgampicillin/mL for E. coli strains containing plasmids other than pGEMderivatives, 100 μg ampicillin/mL for E. coli strains containing pGEMderivatives, or 20 μg kanamycin/mL. R. solanacearum was grown at 30′C inBG media (Boucher et al., 1985).

Bioinformatic Analysis of Acinetobacter OTases.

Protein sequences for Acinetobacter specific OTases were analyzed usingNCBI's Basic Local Alignment Search Tool (BLAST) and protein domainsidentified using the Conserved Domain Database for the annotation ofproteins (Marchler-Bauer et al., 2004, Marchler-Bauer et al., 2009,Marchler-Bauer et al., 2011).

Generation of a Strain with an in-Frame Deletion of pilA.

The ΔpilA mutant was constructed, generating an in-frame deletion ofpilA, according to the methodology published previously by our group(Harding et al., 2013). Primer sets 1 and 2 were used and can be foundin the primer table in the supplemental materials.

Construction of the tfpO::Kan Mutant.

The tfpO::kan mutant was constructed previously (Harding et al., 2013);however, at that time the gene was designated as the pgyA gene, not thetfpO gene.

Complementation of the ΔtfpO::Kan Mutant.

To complement the ΔtfpO::kan mutant, the tfpO gene was cloned with thepredicted pilA promoter into a mini-Tn7 element as previously described.Briefly, gDNA from the ΔpilA mutant was used as template with primer set3 to generate an amplicon containing the predicted pilA promoter, theATG start codon of the pilA open reading frame, a FLP scar, the last 21bp of pilA, the 53 bp intergenic region, and the entire tfpO openreading frame. A four-parental mating strategy was used to introduce themini-Tn7 element containing tfpO driven off the predicted pilA promoterinto the ΔtfpO::kan mutant as previously described (Harding et al.,2013). A correct clone was verified by sequencing and designated as thetfpO+ complement.

Plasmid Construction and Transfer into E. coli.

The pilA gene from strain M2 was PCR amplified from gDNA using primerset 4 and cloned into the EcoRV site of pWH1266. A correct clonecontaining the pWH-pilAM2 plasmid was verified by restriction digestionand sequencing. pWH-pilAM2 was purified from E. coli harboring theplasmid, then electroporated into Acinetobacter isolates. Acinetobacterisolates were made electrocompetent according to the methods previouslydescribed (Aranda et al., 2010). The pBAV-comP-His plasmid was built byusing primer set 32. The sticky-ended amplicon was digested with therespective restriction enzymes and ligated into the vector pBAVmcs inthe same sites. The ligation was then electroporated into DH5α-E withtransformants being selected for on L-agar plates supplemented withkanamycin. The pEXT-pglL_(ComP), pEXT-pglL_(ADP1), pEXT-tfpO 19606 andpEXT-pglL19606 plasmids were built using primer sets 33, 34, 35 and 36respectively. The resulting amplicons were digested with BamHI and SalIand inserted in the same sites of pEXT20. Ligations were electroplatedinto DH5α-E and transformants were selected for on L-agar platessupplemented with ampicillin.

pWH-pilA17978-His Construction and Western Blot Analysis.

The piIA allele from A. baumanii ATCC 17978 was PCR amplified usingprimer set 37. The PCR product was purified and End-it repaired tophosphorylate the 5′ ends. The vector pWH1266 was linearized with EcoRI,End-it repaired, and then treated with alkaline phosphatase. Thelinearized pWH1266 and the PCR purified pilA allele were ligated,transformed into DH5α, and transormants were selected on L-agarsupplemented with ampicillin. The pWH-pilA17978 plasmid was sequencedverified and used as template for an inverse PCR to add a C-terminalhexa-histidine tag using primer set 38. The PCR product was purified,DpnI treated, End-it repaired, and ligated. The ligation waselectroporated into DH5a and transformants were selected on L-agarsupplemented with ampicillin. The pWH-pilA17978-His electroporated intoA. baumannii strains as previously described and naturally transformedinto A. baylyi strains according to our previously described methods fortransforming A. nosocomialis strain M2. Acinetobacter transformants wereselected on L-agar supplemented with ampicillin. Western blot analysison whole cell lysates was conducted as described above with thefollowing exceptions. Strains were grown in LB broth without NaCl andwere normalized to an OD600=2.0.

Construction of a strain M2 ΔpglL::kan mutant. The entire pilA-tfpO-pglLlocus along with 1 kb of flanking DNA from A. nosocomialis strain M2 wasamplified using primer set 6. The PCR product was ligated to pCC1(Epicentre) and transformed into E. coli EPI300. A correct clonecontaining the pCC1-pilA-tfpO-pglL vector was verified by restrictiondigestion and sequencing. To replace the pglL gene with a kanamycincassette, a modified recombineering protocol was used as previouslydescribed (Harding et al., 2013). To introduce the mutation into A.nosocomialis strain M2, the plasmid pCC1-Δpgl::kan was linearized andtransformed via natural transformation. Transformants were selected onL-agar supplemented with kanamycin. The M2ΔpglL::kan region in themutant was verified by sequencing.

Generation of Strains Containing Point Mutations in pilA in Strain M2.

To generate a strain with a carboxy-terminal serine to alanine pointmutation in the pilA gene of strain M2, the M2ΔpilA mutant wascomplemented with a mini-Tn7 element containing a variant of the pilAallele, where the carboxy-terminal serine was mutated to an alanine(pRSM3510-pilA[S136A]). The pRSM3510-pilA[S136A] plasmid was constructedusing the Quikchange Site Directed Mutagenesis Kit (Stratagene)according to the manufacturer's protocol using primer set 8. A correctclone carrying the pRSM3510-pilA[S136A] plasmid was verified byrestriction digest and sequencing. The mini-Tn7 construct containing thepilA[S136A] allele was transposed into the attTn7 of strain M2 via afour-parental mating strategy previously described above. The sameprotocol was used to generate the pilA[S132A] except primer set 39 wasused.

Construction of Glycosyl-Transferase Mutants in the Strain M2Background.

In order to replace each glycosyl-transferase gene with a kanamycinresistance cassette, an In-Fusion HD EcoDry cloning kit was usedaccording to the manufacturers protocol (ClonTech). The followingprotocol describing the construction of the M2ΔweeH::kan mutation wasused for each glycosyl-transferase mutant, except the 15 bp overhangswere added to the primers which amplified the 5′ and 3′ flanking regionsof each respective gene. Briefly, the upstream and downstream flankingDNA regions around weeH were PCR amplified with primer sets 9 and 10respectively. The Tn5 kanamycin cassette and pGEM vector were PCRamplified with 15 bp overhangs homologous to the DNA in which they wereto be recombined using primer sets 11 and 12 respectively. The PCRamplicons were gel extracted and ethanol precipitated. One hundrednanograms of each product was added to the In-fusion EcoDry cloning tubeaccording to the manufacturer's protocol and incubated at 37° C. for 15mins then at 50° C. for 15 mins. The newly generated vector wastransformed into chemically competent Stellar cells (Clontech) accordingto the manufacturer's protocol. Transformants were selected for onL-agar plates supplemented with kanamycin. A correct clone containingthe pGEM-weeH::kan plasmid was sequence verified. The weeH::kan cassettewas PCR amplified using the forward primer of primer set 9 with reverseprimer of primer set 10. The amplicon was DpnI treated, ethanolprecipitated, then transformed into strain M2 according to previouslypublished methodologies. A correct clone designated M2ΔweeH::kan wassequence verified.

The upstream and downstream regions of wafY were amplified using primersets 17 and 18, linearized pGEM was amplified using primer set 15, andthe kanamycin cassette was amplified with primer set 16. The aboveprotocol was used to In-Fuse all four PCR products. pGEM-wafY::kan waslinearized with EcoRI and introduced into A. nosocomialis strain M2 vianatural transformation as previously described. A correct clonedesignated M2 wafY::kan was sequence verified.

The upstream and downstream regions of wafZ were amplified using primersets 19 and 20 and the upstream and downstream regions of wagB wereamplified using primer sets 21 and 22. Mutants were then constructed asdescribed for the wafY mutant. Clones designated M2Δ wafZ::kan and M2wagB::kan were identified and sequence verified.

Constructidon of pRSM4063.

To generate the pRSM4063 vector, an empty mini-Tn7 element was firstintroduced into strain M2 via a four-parental mating strategy previouslydescribed. Transposition of the empty mini-Tn7 element into the attTn7was sequence verified generating the strain M2attTn7::MCS_Empty. GenomicDNA was purified from this strain and used as a template in a PCR usingprimer set 13. The forward primer of primer set 13 is approximately 2 kbupstream of the mini-Tn7 element and the reverse primer of primer set 13is approximately 2 kb downstream of the mini-Tn7 element. The ensuingPCR product was ligated into the pSMART-LCKan vector, sequence verified,and designated pRSM4063.

Constructidon of the weeH Complemented Mutant.

To complement the weeH::kan mutant, weeH plus 375 bp of upstream DNA wasPCR amplified using primer set 14. The amplicon was digested with XmaIand KpnI, cloned into pRSM4063 and electroporated into DH5α. Tocomplement the ΔweeH::kan mutant, pRSM4063-weeH was linearized with NdeIand introduced into M2 ΔweeH::kan via natural transformation accordingto a previously published procedure (Harding et al., 2013).

Construction of the wafY, wafZ, and wagB Complemented Mutants.

Given the lack of an obvious promoter driving expression of each of theglycosyl-transferase genes and the over-lapping nature of the openreading frames, the upstream wxy promoter was selected to driveexpression of each of the three genes. Each glycosyl-transferase mutantwas complemented by returning the deleted gene driven off the predictedwxy promoter to the chromosome using the mini-Tn7 system. Briefly, theglycosyl-transferase locus was PCR amplified using primer set 23, End-Itrepaired (Epicentre) and ligated into pCC1 (Epicentre). Transformantswere selected on chloramphenicol and the pCC1-GT plasmid was verified byrestriction digest. The pCC1-GT plasmid contained the predicted promoterof the wxy gene (329 bp upstream), wxy, wafY, wafZ, wagA, gnaB, andwagB.

To generate the wxy promoter-wafY construct, an inverse PCR strategy wasemployed to remove the wxy gene and join the wxy promoter to the ATGstart codon of the wafY gene using primer set 24 and pCC1-GT astemplate. The subsequent PCR product was End-It repaired (Epicentre) andligated to itself generating the pCC1-wxyP-wafY construct. The wxyP-wafYDNA fragment was PCR amplified using primer set 27, which contained XmaIand KpnI restriction overhangs. The PCR product was digested and ligatedto predigested pRSM3510 then transformed into EC100D cells.Transformants were selected for on L-agar supplemented with kanamycin.The mini-Tn7 element containing wxyPwafY was introduced into theM2ΔwafY::kan mutant using a four-parental mating strategy previouslydescribed.

The same process was used to generate pRSM3510-wxyP-wafY except primerset 25 and primer set 28 were used. The mini-Tn7 element containingwxyP-wafY was introduced to the M2 wafZ::kan mutant via a four-parentalmating strategy. The wxyP-wagB fragment was generated using primer set26 and primer set 29, but was cloned into pRSM4063. ThepRSM4063-wxyP-wagB vector was linearized with XhoI and introduced intoM2ΔwagB::kan via natural transformation as previously described.

Construction and Transfer of p4063-A1S_1193-5× into AcinetobacterStrains.

The A1S_1193 open reading frame along with its predicted promoter wasPCR amplified to include a C-terminal 5× His tag using primer set 30,which also contained XmaI and KpnI restriction overhangs. The PCRproduct was digested and ligated to pre-digested pRSM4063. ThepRSM4063-A1S_193-5× vector was linearized with XhoI and introduced toAcinetobacter strains via natural transformation as previouslydescribed.

Construction of pET-15b-rsPil_(AM2).

A truncated His-tagged recombinant, soluble derivative of pilA(rsPilAM2) was amplified using gDNA from A. nosocomialis strain M2 astemplate with primer set 31 deleting the first 28 amino acids of thePilA protein. This PCR product was then used as template for a secondPCR where the forward primer of primer set 15 contained an NdeI site andthe reverse contained a BamHI site to aid in directional cloning intopET-15b. The amplicon was digested with NdeI and BamHI then ligated intothe expression vector pET-15b, which was digested with NdeI and BamHIgenerating a first codon fusion driven off of the T7 promoter with anN-terminal His tag followed by a thrombin cleavage site. Ligationproducts were electroporated into DH5α-E (Invitrogen), transformantswere subcloned and verified to contain the vector with insert byrestriction digestion and sequencing. A correct clone was transformedinto E. coli strain Origami B (DE3) (Novagen) for expression of therecombinant protein.

rsPilAM2 Purification.

Origami B(DE3) (Millipore) containing pETlSb-rsPilAM2 was inoculatedinto 100 mL of LB broth to an A600 nm optical density of 0.05 and grownat 37° C. with 180 rpm to mid-log phase at which point rsPilAM2expression was induced with IPTG at a final concentration of 500 μM.Cells were transitioned to 19° C. with 180 rpm and grown for 18 h. Cellswere harvested by centrifugation into two equal pellets and resolved in4 mL each of IX Ni-NTA Bind Buffer (Novagen) with protease inhibitors(Roche). Resuspended pellets were added to 15 mL TeenPrep Lysing matrixB tubes (MP Biomedicals) and lysed in a Fast Prep 24 homogenizer (MPBiomedicals) with two rounds at 6.0 m/s for 40 seconds with a 5 minuteincubation on ice between each round. Supernatants were separated fromthe unlysed bacteria and the lysing matrix by centrifugation at 4000 rpmfor 10 mins at 4° C. Supernatants were further clarified with 1 hour ofultracentrifugation at 100,000×g for 1 hour at 4° C. Clarifiedsupernatants were incubated with 1 mL of Ni-NTA His bind resin (Novagen)for 2 hours at 4° C. with gentle rocking followed by two 4 mL washeswith 1×Ni-NTA wash buffer. His-tag rsPilAM2 was eluted from the resinwith three washes of Ni-NTA elution buffer and dialyzed overnight inphosphate buffered saline. The N-terminal His tag on rsPilAM2 wasthrombin cleaved with 0.04 units/μL of biotinylated-thrombin (Novagen)for 2 hours at room temperature. The biotinylated-thrombin was capturedwith streptavidin-agarose beads for 30 minutes and the rsPilA_(M2) wascollected with a centrifugation in a spin filter at 500×g for 5 minutes.To remove any small peptides containing the cleaved His-tag or uncleavedHis-tag rsPilAM2, the filtrate was run over Ni-NTA bind resin and theflow through was collected as pure, cleaved rsPilA_(M2). The pureprotein was dialyzed in phosphate buffer saline with 50% glycerol thennormalized to 1 mg/mL using a BCA total protein assay kit (Pierce).

Generation of Polyclonal Antiserum Against rsPilAM2.

Polyclonal antiserum against rsPil_(AM2) Was raised following ourpreviously described methods (Actis et al., 1985). Briefly, 100 μg ofpurified rsPil_(AM2) emulsified in one milliliter of Freund's completeadjuvant was injected using a 23 gauge needle at ten intracutaneoussites into the haunch of a 6-month old female New Zealand white rabbit(Charles River Laboratories International, Inc., Wilmington, Mass.).Injections consisting of 100 μg rsPil_(AM2) emulsified in Freund'sincomplete adjuvant were subsequently delivered at 15-day intervals, andserum was collected at 10-day intervals following the initial injection.The specificity and reactivity of the anti-rsPil_(AM2) antibodies wereconfirmed by immunoblotting rsPil_(AM2) and A. nosocomialis strain M2whole-cell lysates after proteins were size-fractionated by SDS-PAGE.

Transformation Efficiency Assays.

Natural transformation was assayed as described previously (Harding etal., 2013). Transformation efficiency was calculated by dividing the CFUof transformants by the total CFU. Experiments were conducted on atleast three separate occasions.

Quantitative Dimethylation of A. baylyi ADP1 Membrane Extracts.

Quantitative dimethylation of lysates from A. baylyi ADP1, theADP1ΔpglL_(ComP) mutant, and the ADP1 pglL_(ADP1) mutant was performedas outlined previously (Boersema et al., 2009). Briefly, 1 mg of peptidelysate from each strain was resuspended in 30 μl of 100 mMtetraethylammonium bromide and mixed with the following combinations of200 mM formaldehyde (30 μl) and 1M sodium cyanoborohyride (3 μl)isotopologues: ADP1 samples were labeled with light formaldehyde (CH₂O)and light sodium cyanoborohyride (NaBH₃CN), ADP1ΔpglL_(ADP1) sampleswith medium formaldehyde (CD₂O) and light sodium cyanoborohyride, andADP1Δ pglL_(ComP) with heavy formaldehyde (¹³CD₂O) and heavy sodiumcyanoborodeuteride (NaBD₃CN). Reagents were mixed and samples incubatedat room temperature for 1 h. Dimethylation reactions were repeated twiceto ensure complete labeling of all amine groups. Dimethylation reactionswere terminated by the addition of 30 μl of 1M NH₄Cl for 20 minutes atroom temperature. Samples were acidified by addition of 5% (v/v) aceticacid and allowed to equilibrate in the dark for 1 h before pooling thethree samples at 1:1:1 ratio. Pooled samples were then STAGE tippurified, lyophilized, and stored at −20° C.

Western Blot Analyses.

Western blot analyses were preformed according to previously describedmethodologies (Harding et al., 2013). Primary antibodies used wereAnti-rsPilAM2 or Penta-His Antibody (Qiagen). Secondary antibodies usedwere Goat anti-rabbit IgG (H+L), alkaline phosphatase antibody(Molecular Probes) and Goat-anti mouse IgG (H+L), alkaline phosphataseantibody (Molecular Probes). Membranes were developed with the BCIP/NBTLiquid Substrate System (Sigma).

Pili Shear Preparations.

Pili shear preparations were prepared as previously described with thefollowing modifications. Briefly, bacterial lawns were removed from theagar surface and resuspended in 5 mL of ice cold DPBS supplemented withIX protease inhibitors (Roche). The bacterial suspensions werenormalized to an optical density at A600 nm equal to 70. To shearsurface exposed proteins, bacterial suspensions were vortexed on highfor 1 minute. Bacteria were pelleted at 10,000×g for 10 minutes at 4′C.The supernatants were collected and again centrifuged at 10,000×g for 10minutes at 4′C. The supernatants were collected and further clarified bycentrifugation at 20,000×g for 5 mins at 4′C. The sheared surfaceproteins were precipitated with ammonium sulfate at a finalconcentration of 30%. Precipitated proteins were pelleted bycentrifugation at 20,000×g for 10 minutes at 4′C. The supernatants werediscarded and the pellets were resuspended in 100 μL of 1× Laemmlibuffer. Preparations were boiled for 10 minutes, run on SDS-PAGE,coomassie-stained, and bands were excised and prepared for massspectrometric analysis according to Shevchenko et al. (2006). Briefly,bands were washed with water and dehydrated with acetonitrile (ACN)repeatedly. Disulfide bonds were reduced with 10 mM DTT in 50 mM NH4HCO3for 60 minutes at 37° C. followed by alkylation of cysteine thiol groupswith 50 mM iodoacetamide in 50 mM NH4HCO3 for 60 minutes in the dark atroom temperature. Gel pieces were then washed with 50 mM NH4HCO3,dehydrated with 100% ACN and dried. Pilin was digested with 0.02 mg/mLtrypsin in 50 mM NH4HCO3 (Promega) at 37° C. for 16 hours. Peptides wereeluted with 100% ACN and water and lyophilized. Tryptic peptides wereresuspended in 0.1% Trifluoroacetic acid and desalted with a C18 ZipTip(Millipore, USA). 60% ACN was used to elute the peptides, which weredried in a speedvac and resuspended with 0.1% Formic Acid. The analysiswas done using a Q-TOF Premier (Waters, Manchester, UK) coupled to ananoACQUITY (Waters) ultra-performance liquid chromatography system aspreviously described (Wang et al., 2007). MassLynx, v. 4.1 (Waters) wasused to analyze the data. OmpA-His was purified from strain M2 andprepared for ESI-QTOF MS/MS analysis as described above.

LPS Extraction and Silver Staining

LPS from A. baylyi and R. solanacearum was extracted from overnightcultures by the TRI-reagent method as described previously (Yi &Hackett, 2000). Equal amounts of LPS were loaded on 12.5% SDS-PAGE gelsfor LPS separation followed by silver staining as previously described(Tsai & Frasch, 1982).

DsbA1 Glycosylation in E. coli

E. coli CLM24 cells were co-transformed with three plasmids: one plasmidencoding the C. jejuni glycosylation locus, another plasmid encoding asingle O-OTase gene, and the last plasmid, pAMF22, encoding dsbA1-His.Ampicillin (100 μg/ml), trimethoprim (50 μg/ml) and chloramphenicol (10μg/ml) were added as required for plasmid selection. Cells were grown at37° C. to an OD600 of 0.4-0.6 and then were induced with 0.1 mM IPTGand/or 0.2% arabinose. Cultures requiring arabinose induction were givena second dose of induction after 4 hours. Whole cell lysates wereobtained at stationary phases and western blot analyses were employed todetermine DsbA1 modification.

Digestion of Membrane Enriched Samples of A. baylyi ADP1

Peptide lysates for glycopeptide enrichment and quantitative analysiswere prepared according to Lithgow et al. (2014) with minormodifications. Briefly, 2 mg of dried membrane enriched protein sampleswere solubilized in 6 M urea, 2 M thiourea, 40 mM NH4HCO3 and reducedwith 10 mM Dithiothreitol (DTIT). Reduced, solubilized peptides werealkylated with 25 mM iodoacetamide (IAA) for one hour in the absence oflight. The resulting alkylated protein mixture was digested with Lys-C(1/100 w/w) for 4 hours at 25° C., diluted 1:5 in 40 mM NH4HCO3, thendigested with trypsin (1/50 w/w) overnight at 25° C. Digestion wasterminated with the addition of 1% trifluoroacetic acid (TFA). Peptidedigests were purified using the C18 empore (Sigma-Aldrich, St. LouisMo.) STop And Go Extraction (STAGE) tips (Rappsilber et al., 2007) toremove primary amide and salts.

Enrichment of A. baylyi ADP1 Glycopeptides Using ZIC-HILIC Purification

ZIC-HILIC enrichment was performed according to (Scott et al., 2011)with minor modifications. Micro-columns composed of 10 μm ZIC-HILICresin (Sequant, Umea, Sweden) packed into p10 tips containing a 1 mm²excised C8 Empore™ disc (Sigma) were packed to a bed length of 0.5 cm.Prior to use, the columns were washed with ultra pure water, followed by95% acetonitrile (ACN), and then equilibrated with 80% ACN and 5% formicacid (FA). Samples were resuspended in 80% ACN and 5% FA and insolublematerial was removed by centrifugation at 16,100×g for 5 min at 4° C.Samples were adjusted to a concentration of 3 μg/μL and 150 μg ofpeptide material was loaded onto a column and washed with 10 loadvolumes of 80% ACN, 5% FA. Unbound fractions were collected, pooled, anddried by vacuum centrifugation. ZIC-HILIC bound peptides were elutedwith 3 load volumes of ultra-pure water and concentrated using vacuumcentrifugation. Biological replicates were subjected to ZIC-HILICenrichment independently using freshly prepared reagents.

Identification of Glycopeptides Using Reversed-Phase LC-MS, CID MS-MSand HCD MS-MS

Purified glycopeptides/peptides were resuspended in Buffer A (0.5%acetic acid) and separated using reversed-phase chromatography on eitheran Agilent 1290 Series HPLC (Agilent Technologies, Mississauga, ON)coupled to LTQ-Orbitrap Velos (Thermo Scientific, San Jose Calif.) forqualitative analysis of glycopeptides or an EASY-nLC 1000 system coupledto a Q-exactive for quantitative studies. For qualitative analysis of A.baylyi ADP1 glycopeptides, a packed in-house 20 cm, 75 μm innerdiameter, 360 μm outer diameter, ReproSil—Pur C18 AQ 1.9 μm (Dr. Maisch,Ammerbuch—Entringen, Germany) column was used. For quantitative studiesa house packaged 45 cm, 50 μm inner diameter, 360 μm outer diameter,ReproSil—Pur C18 AQ 1.9 μm column was used. In both systems, sampleswere loaded onto a trap column, an in-house packed 2 cm, 100 μm innerdiameter, 360 μm outer diameter column containing Aqua 5 μm C18(Phenomenex, Torrance, Calif.), at 5 μL/min prior to gradient separationand infused for mass spectrometry. A 180 min gradient was run from 0%buffer B (80% ACN, 0.5% acetic acid) to 32% B over 140 min, next from32% B to 40% B in the next 5 min, then increased to 100% B over 2.5 minperiod, held at 100% B for 2.5 min, and then dropped to 0% B for another20 min. Unbound fractions from ZIC-HILIC glycopeptide enrichment weresubjected to analysis using the same instrumental set up as qualitativeanalysis of glycopeptides. Both instruments were operated using Xcaliburv2.2 (Thermo Scientific) with a capillary temperature of 275° C. in adata-dependent mode automatically switching between MS, CID MS-MS andHCD MS-MS for qualitative analysis as previously described (Scott etal., 2011) and using a top 10 data-dependent approach switching betweenMS (resolution 70 k, AGC target of 1×10⁶), and HCD MS-MS events(resolution 17.5 k AGC target of 1×10⁶ with a maximum injection time of60 ms, NCE 28 with 20% stepping) for quantitative studies.

Identification of A. baylyi ADP1 Glycopeptides

Raw files for qualitative glycosylation analysis were processed aspreviously described (Scott et al., 2011, 2012). Briefly, ProteomeDiscoverer v. 1.2 (Thermo Scientific) was used to search the resultingglycopeptide data using MASCOT v2.4 against the A. baylyi ADP1 database(obtained from UNIPROT, http://www.uniprot.org/, 681 2014-06-10, Taxonidentifier: 62977 containing 3263 protein sequences). Mascot searcheswere performed using the following parameters: peptide mass accuracy 20ppm; fragment mass accuracy 0.02 Da; no enzyme specificity, fixedmodifications—carbamidomethyl, variable modifications—methionineoxidation and deamidated N, Q. The instrument setting of MALDI-QUAD-TOFwas chosen as previous studies show quadrupole-like fragmentation withinHCD spectra (Olsen et al., 2007). Scan events that did not result inpeptide identifications from MASCOT searches were exported to MicrosoftExcel (Microsoft, Redmond Wash.). To identify possible glycopeptideswithin exported non match scans, the MS-MS module of GPMAW 8.2 called‘mgf graph’ was used to identify HCD scan events that contained the204.08 m/z oxonium of HexNAc. All scan events containing the oxonium204.08 m/z ion were manually inspected to identify possibleglycopeptides. To facilitate glycopeptide assignments HCD scan eventscontaining the 204.08 oxonium were manual inspected to identifypotential deglycosylated peptides ions. Within these HCD scans the MSfeatures (m/z, charge and intensity), which corresponded to masses belowthat of the deglycosylated peptide were extracted using the Spectrumlist function of Xcalibur v2.2. The resulting numerical values of thedetected MS features were scripted into mgf files and the peptide massset to that of the deglycosylated peptide mass. The resulting mgf fileswere then searched using the MASCOT setting described above. All spectrawere searched with the decoy option enabled and no matches to thisdatabase were detected; the false discovery rate (FDR) was 0%.

Quantitative analysis of dimethylated A. baylyi ADP1 and mutantglycopeptides was performed as previously reported (Lithgow et al.,2014). Briefly, dimethylated A. baylyi ADP1 glycopeptides wereidentified as above and quantified by manually extracting the area underthe curve of the monoisotopic peak using Xcalibur v2.2. Triplex(wildtype ADP1 vs ADP1 ΔpglL_(ComP) vs ADP1ΔpglL_(ADP1)).

Results

The Two OTase Homologs Encoded by Acinetobacter Contain Different PfamDomains

It was previously reported that A. baylyi ADP1 encodes two proteinscontaining domains from the Wzy_C superfamily (Schulz et al., 2013).Through bioinformatic analyses we identified several Acinetobacter spp.with two open reading frames (ORFs) immediately downstream of the majortype IVa pilin subunit pilA (FIG. 1, panel A) that are predicted toencode proteins that contain evolutionarily related domains from theWzy_C superfamily.

In A. nosocomialis strain M2, the pilA gene is immediately upstream oftwo genes, M215_10480 and M215_10475, both of which encode members ofthe Wzy_C superfamily. M215_10480 and M215_10475 contain the pfam13425and the pfam04932 domains, respectively (FIG. 1, panels A and B). At thetime of this study, the same genetic arrangement was found in 12 of the17 completed genomes for A. baumannii strains, 7 of 8 A. nosocomialisgenomes, and 3 of 5 A. pittii genomes demonstrating the conservation ofthis locus amongst medically relevant members of the Acb complex (Datanot shown). Previously we designated the gene encoding the pfam13425domain containing protein (ORF M215_10480) as the putative glycosylase A(pgyA) (Harding et al., 2013). Given that the gene encoding M215_10480is immediately downstream of pilA, together with the functional dataprovided herein which demonstrates that this protein is a pilinglycosylase, we have renamed the gene encoding ORF M215_10480 as a typefour pilin specific O-Oligosaccharyltransferase gene (tfpO).

The second ORF, M215_10475, encodes a predicted protein that contains adomain from the pfam04932 family, a domain that has been found in allpreviously characterized PglL orthologs as well as in O-antigen ligases.The PglL_A and the PglL_B domains were also identified in M215_10475. Itappears that this protein is an ortholog of the PglL general OTases;thus, we have designated ORF M215_10475 as pglLM2 (FIG. 1, panels A andB).

Transcriptome analysis was performed on mid log phase M2 cells usingRNA-seq (data not shown). PilAM2 transcription was readily observed;tfpOM2 transcription was also observed but the levels droppedprecipitously beginning at the intergenic region between pilAM2 andtfpOM2. A predicted Rho-independent terminator is located immediatelyupstream of the ATG start codon of tfpOM2. It is likely that pilAM2 andtfpOM2 are in an operon separated by a leaky transcriptional terminator.These findings are consistent with transcriptional studies of pilA andpilO (tfpO) of P. aeruginosa 1244 (Castric, 1995). Downstream of thetfpOM2 gene is the pglLM2 gene. Since the genes encoding both OTases arein close proximity and use the same lipid-linked oligosaccharide as asubstrate, it was speculated that these genes would be cotranscribed.There does appear to be some transcriptional read through but transcriptlevels for the pglLM2 gene are markedly higher than the tfpOM2 levels,suggesting that the intergenic region between tfpOM2 and pglLM2 containsa promoter, which also drives transcription of pglLM2.

In the A. baylyi ADP1 genome, the comP gene, encoding a pilin-likeprotein, is followed by ACIAD3337 encoding a pfam04932-containingOTase-like protein, which was designated pglL by Schulz et al., (2013).This has been designated ACIAD3337 as pglL_(ComP) due to its proximityto comP (FIG. 1, panels C and D) and the previously reported evidencedemonstrating its requirement for post-translational modification ofComP (Schulz et al., 2013).

A second pfam13425 domain containing ORF (ACIAD0103) predicted to encodea WaaL ligase ortholog was also identified. ACIAD0103 was not locatednear the pilin gene homolog, but instead was found within a glycanbiosynthetic locus. This is herein designated ACIAD0103 as thepglL_(ADP1) (FIG. 1, panels C and D). A. baylyi was the only straincontaining two genes encoding proteins with domains from the Wzy_Csuperfamily that were not encoded by adjacent genes.

In A. baumannii ATCC 17978, a well-studied strain with respect to itsglycosylation, only one general O-OTase was identified, which waspreviously designated PglL (Iwashkiw et al., 2012; Lees-Miller et al.,2013).

TfpO is required for post-translational modification of pilin in A.nosocomialis strain M2.

FIG. 2A shows Western blot analysis of whole cell lysates from strainM2, the isogenic pilA mutant, and the complemented pilA mutant strainconfirmed our previous findings that PilA existed in two molecular formsdiffering by apparent molecular weight. The more abundant, highermolecular weight form of PilA was likely a post-translationally modifiedspecies of PilA while the lower molecular weight form of PilA was anunmodified species. To determine the effects of TfpO on PilApost-translational modification, we constructed an isogenic tfpO mutantand probed for PilA expression. PilA from the strain lacking tfpOexisted only in the lower molecular weight form (FIG. 2A). The increasein PilA's electrophoretic mobility is consistent with the loss of apost-translational modification. Furthermore, PilA from the complementedtfpO mutant strain existed primarily in the higher molecular weight formconfirming that TfpO was required for post-translational modification ofPilA.

Immediately downstream of tfpO in strain M2 is pglLM2, which encodes ahomolog of the general O-OTases responsible for glycosylation of manymembrane associated proteins in Neisseria gonorrhoeae and N.meningiditis (Vik et al., 2009; Børud et al., 2011). To determine ifPglLM2 also played a role in post-translational modification of PilA, anisogenic mutant strain lacking pglLM2 was generated. Western blotanalysis of whole cell lysates from the pglLM2 mutant demonstrated thatPilA existed primarily in the modified form, indicating that PglLM2 isnot required for the post-translational modification of PilA as observed(FIG. 2A).

PilA_(m2) was Glycosylated in a TfpO_(M2)-Dependent Manner with aTetrasaccharide Containing (HexNAc)₂, Hexose and N-Acetyl-deoxyHexose.

To confirm that PilA was glycosylated by TfpO, PilA was purified fromsurface shear preparations from strain M2, a hyper-piliated M2ΔpilTmutant, and a hyper-piliated M2ΔtfpO::kanΔpilT::strep mutant. The pilTgene encodes for the predicted retraction ATPase; therefore, mutantslacking pilT have a hyper-piliated phenotype, which results in anabundance of surface exposed PilA. Proteins in the shear preparationswere separated by SDS-PAGE, coomassie-stained, excised and subjected tomass spectrometric analysis. FIG. 2B shows MS/MS analysis of PilA fromboth strains M2 and M2ΔpilT identified the presence of atetrasaccharide, comprised of two HexNAc residues, a Hexose andN-acetyldeoxyHexose, on PilA. MS/MS analysis revealed that thetetrasaccharide was present on the carboxy-terminal tryptic119NSGTDTPVELLPQSFVAS 136 peptide. PilA from theM2ΔtfpO::kanΔpilT::strep mutant was unmodified confirming that TfpO wasrequired for PilA glycosylation (data not shown).

The Carboxy-Terminal Serine136 of PilAM2 was Required for PilinModification.

In P. aeruginosa 1244 the pilin protein PilA is glycosylated in aTfpO-dependent manner (Castric, 1995). The glycosylation site was laterdetermined to be at the carboxy terminal serine 148 (Comer et al.,2002). Amino acid sequences of PilA proteins from Acinetobacter spp.,including A. nosocomialis M2, were compared to the P. aeruginosa 1244PilA. Although the sequences share limited homology, strain M2's PilAsequence also contains a C-terminal serine, which was included in theglycopeptide identified by MS (FIG. 2B).

FIG. 3A shows that, in fact, all Acinetobacter spp. containing twoconsecutive genes encoding O-OTase homologs contain a carboxy-terminalserine in their respective PilA amino acid sequences.

In order to determine if the carboxy-terminal serine 136 was requiredfor PilA post-translational modification, the M2(pilA[S136A])+ strainwas generated. First, a strain with an in-frame deletion of the pilAgene was generated so as to not affect the transcription of thedownstream tfpO gene. We then complemented the M2ΔpilA strain with anallele of pilA where the carboxy-terminal serine was mutated to analanine residue generating an M2(pilA[S136A])+ strain.

FIG. 3B shows Western blot analysis of whole cell lysates from theM2(pilA[S136A])+ strain demonstrated that PilA only existed in theunmodified, lower molecular weight form indicating that thecarboxy-terminal serine was required for PilA post-translationalmodification). Another highly conserved serine was found at position132. We constructed the M2(pilA[S132A])+ strain in order to determine ifthis site was also required for glycosylation. Western blot analysis ofwhole cell lysates from the M2(pilA[S132A])+ strain demonstrated thatPilA existed in the modified form indicating that serine 132 was notrequired for glycosylation (FIG. 3B). The carboxy terminal serine toalanine point mutation did not affect Tfp functionality as theM2(pilA[S136A])+ strain was naturally transformable (FIG. 3C) andexhibited twitching motility similar to the parental strain (Data notshown).

The Major Polysaccharide Antigen (MPA) Locus is Required forPost-Translational Modification of PilAM2.

Hu et al. recently developed a molecular serotyping scheme forAcinetobacter spp. containing a major polysaccharide antigen (MPA)locus. The MPA locus, found between the conserved fkpA and lldP genes,was identified in all sequenced Acinetobacter strains included in theirstudy and was also present in A. nosocomialis strain M2 (Hu et al.,2013, Carruthers et al., 2013). The MPA locus from A. nosocomialisstrain M2 contains three predicted glycosyl-transferases (designatedwaft, wafJZ, and wagB) and one predicted initiating glycosyl-transferase(designated weeH orpglC) (FIG. 4A). To determine if the MPA locus wasrequired for post-translational modification of PilAM2, individualisogenic mutants lacking each of the predicted glycosyltransferases wereconstructed. FIG. 4B shows Western blot analysis of whole cell lysatesfrom the strain lacking weeH demonstrated that PilA existed in the lowermolecular weight form indicating that WeeH is required for glycosylationof PilA. Deletion of the other three glycosyltransferases yielded PilAproteins with intermediate electrophoretic mobilities. PilA from thewafY::kan mutant migrated closest to the WT PilA mobility, then PilAfrom the wafZ::kan mutant, followed by PilA from the wagB::kan mutant(FIG. 4B). Interestingly, both partially modified and unmodified formsof PilA were identified from the wafZ::kan and wagB::kan mutantbackgrounds. All mutant strains were successfully complemented,indicating that the products of waft, wafZ, wagB, and weeH genes wereall required to produce fully modified PilA.

pglLM2 Encodes a PglL-Like O-OTase in A. nosocomialis Strain M2 and Usesthe Same Tetrasaccharide Precursor as a Donor for General ProteinGlycosylation.

In FIG. 2A, pglLM2, the second ORF containing the Wzy_C domain, wasshown to not be required for pilin glycosylation. It is thought thatpglLM2 may be a general O-OTase that, like the previously characterizedPglL in A. baumannii ATCC 17978, could glycosylate non-pilin targetproteins. We recently demonstrated that A1S_1193-His, encoding for theprotein OmpA, could serve as a bait acceptor protein in order to isolateand identify Acinetobacter strain specific glycans, as it is recognizedby PglLs from different strains (Scott et al., 2014). We expressedOmpA-His, containing a carboxy terminal His-tag, in strains M2,M2ΔtfpO::kan, M2ΔpglL::kan, and M2ΔweeH::kan.

FIG. 5A provides Western blot analysis which demonstrates that all fourstrains expressed OmpA-His; however, OmpA-His from the M2ΔpglL::kan andthe M2ΔweeH::kan backgrounds migrated at an increased electrophoreticmobility, consistent with the lack of a post-translational modification.ESI-TOF-MS/MS analysis of OmpA-His purified from strain M2 revealedglycosylation with two subunits of a branched tetrasaccharide. Theseresults suggest that M215_10475 is a general O-OTase providingfunctional evidence for the PglLM2 designation. Furthermore, thisbranched tetrasaccharide was the same tetrasaccharide found on PilA,indicating that TfpOM2 and PglLM2 both utilize the same lipid-linkedglycan precursor as the substrate for protein glycosylation (FIG. 5B).This observation was expected given that WeeH was required for both PilAand OmpA-Hispost-translational modification, indicating a common glycanprecursor pathway.

ACIAD0103 is not a WaaL O-antigen lgase and is not required for ComPmodification.

As noted above, two OTase-like proteins containing domains from theWzy_C superfamily are encoded in the A. baylyi ADP1 genome. One ofthese, pglL_(ComP) (ACIAD3337), is located adjacent to comP. Schulz etal. (2013) determined, and we independently confirmed, that pglL_(ComP)(ACIAD3337) is required for ComP modification (FIG. 6A). Furthermore,western blot analysis probing for ComP-His expression from an isogenicpglL_(ADP1) (ACIAD0103) mutant strain demonstrated that PglL_(ADP1) isnot required for ComP post-translational modification (FIG. 6A).

Schulz et al. (2013) speculated that the other Wzy_C superfamilydomain-containing protein, PglL_(ADP1) (ACIAD0103), encoded a WaaLO-antigen ligase. For LPS biosynthesis, the O-antigen repeat unit issequentially assembled on the same lipid carrier as the O-glycan on thecytoplasmic side of the inner membrane, flipped to the periplasm,polymerized to form the O-antigen chain and transferred to the lipidA-core polysaccharide by the O-antigen ligase (Hug & Feldman, 2011).Differences in the number of O-antigen subunit repeats in LPS moleculesappear as a ladder-like banding pattern in LPS silver stains (Whitfield,1995). In order to determine if pglL_(ADP1) was acting as an O-antigenligase, we purified LPS from A. baylyi ADP1, the ADP1ΔpglL_(ComP)::kan,and ADP1ΔpglL_(ADP1)::kan mutants and silver stained the SDSPAGE-separated preparation.

As illustrated in FIG. 6B, LPS silver stained SDS polyacrylamide gelsshowed identical banding patterns, with no O-antigen subunits observedin the LOS compared to O-antigen-containing LPS obtained from the plantpathogen Ralstonia solanacearum. Given that PglL_(ADP1) was not actingas an O-antigen ligase, it is possible that PglL_(ADP1) could encode asecond O-OTase.

ACIAD0103 Encodes the General O-OTase, PglLADP1, in A. baylyi ADP1

PglL_(ADP1) was tested to determine if it is able to glycosylate DsbA1from N. meningitidis and OmpA from A. baumannii ATCC 17978, which arealso modified by general O-OTases in their respective strains, and werepreviously employed as models to study glycosylation (Vik et al., 2009;Iwashkiw et al., 2012; Gebhart et al., 2012; Lithgow et al., 2014).These two proteins were independently expressed in wild-type,ΔpglL_(ComP) and ΔpglL_(ADP1) A. baylyi strains. DsbA1-His (FIG. 6C) andOmpA-His (FIG. 6D) displayed an increased electrophoretic mobility inthe ΔpglL_(ADP1) background relative to wild-type and ΔpglL_(ComP)backgrounds. These experiments support the role of PglL_(ADP1) as ageneral O-OTase.

In vivo glycosylation assays in E. coli were performed to furtherconfirm the OTase activity of PglL_(ADP1) (Gebhart et al., 2012). Weemployed E. coli CLM24, a strain lacking the WaaL O-antigen ligase,which leads to the accumulation of lipid-linked glycan precursors thatthen are able to serve as substrates for heterologous O-OTase activity(Feldman et al., 2005). E. coli CLM24 was transformed with plasmidsencoding an acceptor protein (DsbA1), a glycan donor (the Campylobacterjejuni lipid-linked oligosaccharide (CjLLO)), and one OTase, aspreviously described (Faridmoayer et al., 2007; Ielmini & Feldman,2011). We also included PglL_(ComP) and employed TfpO 19606 andPglL19606, encoding the pilin-specific and the general O-OTase from A.baumannii ATCC 19606, as controls. DsbA1-His was detected with ananti-histidine antibody and glycosylation was detected employing the hR6antibody, which is reactive against the C. jejuni heptasaccharide.

As illustrated in FIG. 7, a band reacting with both antibodies,corresponding to DsbA1-His modified by the C. jejuni heptasaccharide,was only present in E. coli coexpressing DsbA1-His, the CjLLO and eitherPglL_(ADP1) or the general O-OTase PglL19606. Together this datasuggests a role of PglL_(ADP1) as a general O-OTase.

Comparative proteomic analysis of A. baylyi ADP1 wild-type, ΔpglL_(ComP)and ΔpglL_(ADP1) strains.

To determine a role of both putative O-OTases in A. baylyi ADP1glycosylation, we compared the glycoproteome of A. baylyi ADP1 to eitherthe ADP1ΔpglL_(ComP) mutant or the APD1ΔpglL_(ADP1) mutant. UsingZIC-HILIC for glycopeptide enrichment (Iwashkiw et al., 2012; Nothaft etal., 2012; Scott et al., 2014; Lithgow et al., 2014) and multiple MS/MSfragmentation approaches (Scott et al., 2011), 21 unique glycopeptidesfrom eight protein substrates were identified 360 within A. baylyi ADP1.Similar to the diversity observed within other Acinetobacter spp. (Scottet al., 2014), A. baylyi ADP1 generated unique glycans withglycopeptides decorated with one of four pentasaccharide glycoformscomposed of 286-217-HexNAc3 (1112.41 Da, FIGS. 8A and 8D),286-217-245-HexNAc2 (1154.41 Da, FIGS. 8B and 8F),286-217-HexNAc-245-HexNAc (1154.41 Da, FIGS. 8B and 8G) and286-217-2452-HexNAc (1196.41 Da, FIGS. 8C and 8E). Glycopeptide analysisof membrane proteins from A. baylyi ADP1ΔpglL_(ComP) enabled theidentification of identical glycopeptides suggesting the glycoproteomewas unaffected by the loss of this gene. In contrast none of 21glycopeptides observed within wild type A. baylyi ADP1 could be detectedwithin extracts of A. baylyi ADP1ΔpglLADP₁ (data not shown), suggestingthat pglL_(ADP1) was responsible for general protein glycosylationwithin A. baylyi ADP1.

Glycopeptide Quantitative Labeling

FIG. 19 shows quantitative analysis of glycosylation in A. baylyi ADP1WT, A. baylyi ADP1ΔpglL_(ADP1), and A. baylyi ADP1ΔpglL_(ComP) usingdimethyl labeling.

Quantitative dimethylation labeling enabled comparison of all threestrains simultaneously providing an internal positive control forglycopeptide enrichment and led to the detection of seven glycopeptides(Table 2). Consistent with the requirement of PglL_(ADP1) forglycosylation, no glycopeptides derived from the ΔpglL_(ADP1) mutant(FIG. 19 A-C) could be detected, while non-glycosylated peptides withinthe samples were observed at a ˜1:1:1 ratio (FIG. 19 D-E, Table 2).Glycosylation was observed at near 1:1 ratio in ΔpglL_(ComP) compared towild type (ranging from 46% to 170%, FIG. 21) with MS/MS identificationsenabling the confirmation of glycopeptides originated from strainΔpglL_(ComP) (FIG. 19C). Taken together these data suggests PglL_(ADP1)is a general O-linked OTase while PglL_(ComP) is responsible forglycosylation of a specific subset of the glycoproteome, which was notdetectable given the sensitivity of the method employed.

Using dimethyl labeling and ZIC-HILIC, the O-OTase responsible forglycosylation of individual glycopeptides was confirmed. Glycopeptidesderived from A. baylyi ADP1 WT, labeled with light label and A. baylyiADP1ΔpglL_(ComP) labeled with heavy label, were observed at near 1:1levels; whereas, A. baylyi ADP1ΔpglL_(ADP1), labeled with medium label,was undetectable within samples. Conversely non-glycosylated peptideswere observed at a near 1:1:1 level between all three strains. A and D)The MS spectra of the light, medium and heavy isotopologues of theglycopeptide 113KLAEPAASAVADQNSPLSAQQQLEQK138 (SEQ ID NO: 108)(Q6F825_ACIAD) and non-glycosylated peptide 166AQSVANYLSGQVSSSR182 (SEQID NO: 109) (Q6FDR2_ACIAD) enabled the comparison of glycosylationacross all three strains. No glycopeptides were observed withinADP1ΔpglL_(ADP1) while non-glycosylated peptides were observed a near1:1:1 ratio. B and E) Comparison of the extracted ion chromatograms ofthe light, medium and heavy isotopologues confirm the absent ofADP1ΔpglL_(ADP1) derived glycopeptides and the 1:1:1 ratio ofnon-glycosylated peptides. C) HCD fragmentation confirming theidentification of the heavy isotopologues of the glycopeptide113KLAEPAASAVADQNSPLSAQQQLEQK138 (SEQ ID NO: 108), confirming itsorigins from ADP1ΔpglL_(ComP). F) HCD fragmentation confirming theidentification of the medium isotopologues of the non-glycosylatedpeptide 166AQSVANYLSGQGVSSSR182 (SEQ ID NO: 109), confirming its originsfrom ADP1ΔpglL_(ADP1).

Table 2 shows dimethylated glycopeptides identified in A. baylyi ADP1.Dimethylated Glycopeptides identified in A. baylyi ADP1 wild type(light) and OTase mutant (heavy). Identifications are grouped accordingto the corresponding Uniprot number. The protein name, parent m/z,charge state, glycan mass, peptide mass, glycan composition, peptidesequence and mascot ion score are provided for each identifiedglycopeptide. For each identified peptide the dimethylation observed isdenoted to aid read distinguish glycopeptide observed from wild type(containing dimethyl N-term and K) and the complement (containingdimethyl N-term 2H(6)13C(2) and K2H(6)13C(2)).

TABLE 2Dimethylated glycopeptides identified in Acinetobacter baylyi ADP1 (SEQ ID NOs: 79-82)Glycan Precursor Precursor Mascot ion Number of Protein Fasta headerspeptide Charge mass m/z MH+ score labels Q6F7U4 >tr|Q6F7U4|Q6F7U4_ACIADSSELEDLFNSDGGAASEPAASDKTAAK 4 1112.41 966.9369 3864.7238  96Dimethyl (K); Uncharacterized protein Dimethyl (K); OS =Acinetobacter baylyi (strain Dimethyl (N- ATCC 33305/BD413/ADP1) term)GN = ACIAD3186 PE = 4 SV = 1 Q6F825 >tr|Q6F825|Q6F825_ACIADKLAEPAASAVADQNSPLSAQQQLEQK 4 1112.41 980.4813 3918.9013  47Dimethyl (K); Uncharacterized protein Dimethyl (K); OS =Acinetobacter baylyi (strain Dimethyl (N- ATCC 33305/BD413/ADP1) term)GN = ACIAD3092 PE = 4 SV = 1 Q6FCV1 >tr|Q6FCV1|Q6FCV1_ACIADIDAAADHAAASTEHAADKAEVATR 4 1112.41 890.9106 3560.6187 112 Dimethyl (K);Uncharacterized protein Dimethyl (N- OS = Acinetobacter baylyi (strainterm) ATCC 33305/BD413/ADP1) GN = ACIAD1233 PE = 4 SV = 1Q6F8B6 >tr|Q6F8B6|Q6F8B6_ACIAD SASKPNVEASVSSQNATLSASQPQHQ 4 1154.43966.6921 3863.7684  52 Dimethyl (K); Uncharacterized proteinDimethyl (N- OS = Acinetobacter baylyi (strain term)ATCC 33305/BD413/ADP1) GN = ACIAD2990 PE = 4 SV = 1Q6FCV1 >tr|Q6FCV1|Q6FCV1_ACIAD IDAAADHAAASTEHAADKAEVATR 4 1154.43901.4081 3602.6325 122 Dimethyl (K); Uncharacterized proteinDimethyl (N- OS = Acinetobacter baylyi (strain term)ATCC 33305/BD413/ADP1) GN = ACIAD1233 PE = 4 SV = 1Q6F8B6 >tr|Q6F8B6|Q6F8B6_ACIAD SASKPNVEASVSSQNATLSASQPQHQ 4 1196.43977.1939 3905.7756  39 Dimethyl (K); Uncharacterized proteinDimethyl (N- OS = Acinetobacter baylyi (strain term)ATCC 33305/BD413/ADP1) GN = ACIAD2990 PE = 4 SV = 1Q6FCV1 >tr|Q6FCV1|Q6FCV1_ACIAD IDAAADHAAASTEHAADKAEVATR 4 1196.43911.9099 3644.6396 118 Dimethyl (K); Uncharacterized proteinDimethyl (N- OS = Acinetobacter baylyi (strain term)ATCC 33305/BD413/ADP1) GN = ACIAD1233 PE = 4 SV = 1(SEQ ID NOs: 79-82)

FIG. 21 (SEQ ID NOs: 79-82) illustrates quantitative glycopeptidesidentified in A. baylyi ADP1. Dimethylated glycopeptides identified inA. baylyi ADP1 wild type (light) and OTase mutant (heavy).Identifications are grouped according to the corresponding Uniprotnumber. The protein name, parent m/z, charge state, glycan mass, peptidemass, glycan composition, peptide sequence and mascot ion score areprovided for each identified glycopeptide. For each identified peptidethe dimethylation observed is denoted to aid read distinguishglycopeptide observed from wild type (containing dimethyl N-term and K)and the mutant (containing dimethyl N-term 2H(6)13C(2) and K2H(6)13C(2).

PglL_(ComP) Acceptor Protein Specificity Distinguishes it from TfpO andGeneral O-OTases

Given the strong genetic linkage between the major type IVa pilin genesand downstream O-OTase genes on Acinetobacter chromosomes, we sought todetermine whether these O-OTases were specific for their cognate pilinprotein. A plasmid expressing PilAM2 from A. nosocomialis strain M2 wasexpressed into different Acinetobacter spp., and then conducted westernblot analysis probing for the expression and electrophoretic mobility ofthe pilin protein. PilAM2 was modified by A. baumannii ATCC 19606 andthe clinical isolate A. baumannii 27413, both of which encode tfpOhomologs and pilins containing terminal serine residues, as evidenced bythe presence of both the higher molecular weight and lower molecularforms of PilAM2 (FIG. 9A). The glycan associated with A. baumannii ATCC19606 was demonstrated to be identical to the pentasaccharide identifiedin A. baumannii ATCC 17978. PilAM2 expressed in A. baumannii ATCC 19606ran with the slowest electrophoretic mobility indicative of a largerglycan associated with PilAM2 (Scott et al., 2014). Both A. baumanniiATCC 17978 and A. baylyi ADP1, which lack a tfpO homolog, were unable toglycosylate PilAM2.

On the contrary, when ComP-His was heterologously expressed in differentAcinetobacter spp., it was found that it was glycosylated only in A.baylyi ADP1. Strains encoding tfpO homologs were unable to modifyComP-His, with the exception of A. baumannii ATCC 19606, which appearedto have a marginal capacity to modify ComP-His (FIG. 9B). PglL_(ComP)was analyzed to determined if it is able to modify A. baumannii ATCC17978 pilin, which does not carry a terminal serine residue. PilA17978was not glycosylated by PglL_(ComP), but was glycosylated by both itscognate PglL17978 and the PglL_(ADP1) general O-OTases. These resultsdistinguish PglL_(ComP) from the other pilin-specific TfpO OTases thatrecognize terminal serine residues, and from the general PglL O-OTases.The sequence of PglL_(ComP) is more similar to PglL-like OTases but,unexpectedly, its activity is specific for ComP, which is a pilin-likeprotein that does not have a terminal serine residue. PglL_(ComP)appears to belong to a new class of O-OTases.

Multiple species within the Acinetobacter genus are nosocomialopportunistic pathogens of increasing relevance worldwide. Among thevirulence factors utilized by these bacteria are the type IV pili and aprotein O-glycosylation system. Glycosylation is mediated byO-oligosaccharyltransferases (O-OTases), enzymes that transfer theglycan from a lipid carrier to target proteins. O-OTases are difficultto identify due to similarities with the WaaL ligases that catalyze thelast step in LPS synthesis. A bioinformatics analysis revealed thepresence of two genes encoding putative O-OTases or WaaL ligases in mostof the strains within the genus Acinetobacter. Employing A. nosocomialisM2 and A. baylyi ADP1 as model systems, the present application providesthat these genes encode two O-OTases, one devoted uniquely to type IVpilin, and the other one responsible for glycosylation of multipleproteins. With the exception of ADP1, the pilin-specific OTases inAcinetobacter resemble the TfpO/PilO O-Otase from Pseudomonasaeruginosa. In ADP1 instead, the two O-OTases are closely related toPglL, the general O-OTase first discovered in Neisseria. However, one ofthem is exclusively dedicated to the glycosylation of the pilin-likeprotein ComP.

Glycopeptides identified in Acinetobacter baylyi ADP1 wild type areshown in Table 3. Identifications are grouped according to thecorresponding Uniprot number. The protein name, parent m/z, chargestate, glycan mass, peptide mass, glycan composition, peptide sequenceand mascot ion score are provided for each identified glycopeptide.

Table 3: Glycopentides Identified in A. Baylyi ADP1 (SEO ID NOs: 79-88)

TABLE 3 Glycopeptides identified in Acinetobacter baylyi ADP1 ProteinPre- Pre- Pre- accession cursor cursor cursor RT Mascot Peptide Glycannumber Protein name m/z [Da] MH+ [Da] Charge [min] Peptide score massmass page Q6F875_ Uncharacterized  703.98 2109.92 3 19.49 AAHAASAAASK 62 955.50 1154.43  3 ACIAD protein Q6F875_ Uncharacterized  717.98 2151.933 20.80 AAHAASAAASK 35  955.50 1196.43  4 ACIAD protein Q6FCV1_Uncharacterized  766.33 2296.97 3 29.47 DAAHDAAASVEK 54 1184.56 1112.41 5 ACIAD protein Q6FCV1_ Uncharacterized  720.06 2877.23 4 27.44IDAAADHAAASTEHAADK 40 1764.82 1112.41  6 ACIAD protein; Q6FCV1_Uncharacterized  730.57 2919.24 4 27.93 IDAAADHAAASTEHAADK 32 1764.821154.42  7 ACIAD protein; Q6FCV1_ Uncharacterized  741.07 2961.25 428.51 IDAAADHAAASTEHAADK 46 1764.82 1196.44  8 ACIAD protein; Q6FCV1_Uncharacterized  701.72 3504.57 5 37.64 IDAAADHAAASTEHAADKAEVATR 462392.15 1112.42  9 ACIAD protein; Q6FCV1_ Uncharacterized  710.123546.58 5 38.00 IDAAADHAAASTEHAADKAEVATR 38 2392.15 1154.43 10 ACIADprotein; Q6FCV1_ Uncharacterized 897.90 3588.59 4 39.70IDAAADHAAASTEHAADKAEVATR 21 2392.15 1196.44 11 ACIAD protein; Q6F7K5_Uncharacterized 1579.39 4736.14 3 83.54IYQNTDTSSAASQTSASPTTQGLGDFLHAQEQLR 23 3623.72 1112.42 12 ACIAD proteinQ6F7K5_ Uncharacterized 1593.39 4778.15 3 84.29IYQNTDTSSAASQTSASPTTQGLGDFLHAQEQLR 47 3623.72 1154.43 13 ACIAD proteinQ6F7K5_ Uncharacterized 1607.39 4820.16 3 84.77IYQNTDTSSAASQTSASPTTQGLGDFLHAQEQLR 50 3623.72 1196.44 14 ACIAD proteinQ6F825_ Uncharacterized 1278.95 3834.82 3 55.92KLAEPAASAVADQNSPLSAQQQLEQK 50 2722.40 1112.42 15 ACIAD protein Q6F825_Uncharacterized 1292.95 3876.83 3 55.87 KLAEPAASAVADQNSPLSAQQQLEQK 582722.40 1154.43 16 ACIAD protein Q6F825_ Uncharacterized  980.46 3918.844 55.74 KLAEPAASAVADQNSPLSAQQQLEQK 48 2722.40 1196.44 17 ACIAD proteinQ6F814_ Putative  814.04 2440.11 3 20.96 NTAASSVAATHKK 65 1285.691154.42 18 ACIAD secretion protein (HlyD family) Q6F814_ Putative 828.05 2482.12 3 20.59 NTAASSVAATHKK 56 1285.69 1196.44 19 ACIADsecretion protein (HlyD family) Q6F8B6_ Uncharacterized 1283.91 3849.723 37.41 SASKPNVEASVSSQNATLSASQPQHQ 57 2653.28 1196.44 20 ACIAD proteinQ6F7U4_ Uncharacterized 1260.88 3780.64 3 66.46SSELEDLFNSDGGAASEPAASDKTAAK 60 2668.22 1112.41 21 ACIAD protein Q6FAJ2_Uncharacterized  989.48 3954.88 4 66.33 VEQIVAQPAPASSVQFKPSNPEIDYK 262842.46 1112.42 22 ACIAD protein Q6FAJ2_ Uncharacterized  999.98 3996.894 66.92 VEQIVAQPAPASSVQFKPSNPEIDYK 21 2842.47 1154.43 23 ACIAD protein

EXAMPLE 2

Materials and Methods:

Strains and Plasmids

A list of the bacterial strains and plasmids used in this study is foundin Table 4. A. baylyi genomic DNA was isolated using the DNeasy bloodand tissue kit (Qiagen). For amplifying the gene aciad3337 (pglL_(ComP))with its upstream non-coding region, primers igrF(ACTGGTCGACTAGTAGTACTATATGGCTITAAA; SEQ ID NO: 89) and igrR(ACTGCTGCAGITAATATCTAITGAACAAAATIITAAC; SEQ ID NO: 90) were used. Theresulting PCR product was digested with SalI and PstI and inserted inthe same sites of pEXT20, creating pMN8. Plasmid pMN1 was constructed bysubcloning ComP from pBAV-ComP-his into BamHI and SalI sites of pEXT20.Plasmid pMN2 was constructed by subcloning pglL_(ComP) with its upstreamregion from the SalI and PstI sites of pMN8 to pMN1.

TABLE 4 Bacterial Strains and plasmids used in this study Reference/Strain/Plasmid Description Source Strains E. coli CLM24 W3110, ΔwaaLligase Feldman et al. (2005) E. coli CLM37 W3110, ΔwecAglycosyltransferase Linton et al. (2005) E. coli SDB1 W3110, Δ waaLligase, ΔwecA glycosyltransferase Garcia- Quintanilla et al. (2014) E.coli DH5α General cloning strain Invitrogen Plasmids pEXT20 Cloningvector, Amp^(R), IPTG inducible Dykxhoorn et al., (1996) pBAV-ComP-hisC-6X His-tagged ComP cloned in BamHI and SalI sites Harding et ofpBAVmcs, Kan^(R), constitutive expression al., (2015) pMN1 C-6XHis-tagged ComP cloned in BamHI and SalI sites This work of pEXT20,Amp^(R), IPTG inducible pMN2 Non-coding region and PglL_(ComP) cloned inSalI and PstI This work sites of pMN1, Amp^(R), IPTG inducible pMN3 ComPS69A in pMN2 background This work pMN4 PglL_(ComP) H325A in pMN2background This work pMN8 Non-coding region and PglL_(ComP) cloned inSalI and PstI This work sites of pEXT20, Amp^(R), IPTG inducible pMAF10HA-tagged PglB cloned in pMLBAD, TpR, Arabinose Feldman et inducible al.(2005) pAMF10 C-10× His-tagged NmPglL cloned inyo pEXT20, FaridmoayerAmpR, IPTG inducible et al., (2008) pIH18 C-6X His-tagged AcrA from C.cloned into pEXT21, Hug et al., SpR, IPTG inducible 2010 pAMF22 C-6XHis-tagged dsbA1 from N. meningitidis MC58 Faridmoayer cloned intopMLBAD, Tp^(R) Arabinose inducible et al., (unpublished) pACYCpglBmutpACYC184-based plasmid encoding the C. jejuni pgl Wacker et locus withmutations W458A and D459A in PglB. Cm^(R), al., (2002) IPTG inducible.pEXT20-pglL_(ADP1) pEXT20 expressing C-6X His-tagged pglLADP1 fromHarding et A. baylyi inserted at BamHI and SalI, Amp^(R), IPTG al.,(2015) inducible pLPS2 P. aeruginosa O11 antigen synthesis cluster,Tet^(R) Goldberg et al., (1992) pJHCV32 E. coli O7 antigen synthesiscluster, Tet^(R) Feldman et al, (1999) pNLP80 S. pneumoniae CPS14cluster on pWSK129, Kan^(R) Price et al., unpublished pOSH59 S.pneumoniae CPS15b cluster on pACT3, Cm^(R) Posch et al., unpublishedpPR1347 S. enterica serovar Typhimurium O antigen synthesis Neal et al.,cluster, Kan^(R) (1993)

Glycosylation in E. coli

Electrocompetent E. coli CLM24, CLM37 or SDB1 were prepared as per theprotocol described by Dower and colleagues (Dower et al., 1988). Cellswere transformed with plasmids encoding the glycan synthesis loci,acceptor protein and OTase. For plasmid selection, ampicillin (100μg/ml), tetracycline (20 μg/ml), trimethoprim (50 μg/ml),chloramphenicol (12.5 μg/ml), kanamycin (20 μg/ml) and spectinomycin (80μg/ml) were added as needed. Cells were grown at 37° C. in LB broth toan OD₆₀₀ of 0.4-0.6, induced with 0.05 mM or 0.1 mM IPTG or 0.2%arabinose as required and left overnight at 37° C. Cultures requiringarabinose induction received a second dose of arabinose after 4 hours.Whole cell lysates were obtained at stationary phases, of which 0.1 ODwere loaded on 12.5% SDS-PAGE gels, which were then transferred tonitrocellulose membranes. Western blot analysis was employed todetermine protein modification and antibodies used are outlined in Table5. Nitrocellulose membranes were visualized using the Odyssey InfraredImaging System (LiCor Biosciences, USA).

TABLE 5 Antibodies used in the present application Reference/ AntibodyDescription Source Anti His Poly Rabbit polyclonal, 1:4000 Rockland AntiHis Mono Mouse monoclonal, 1.5000 Abcam Anti CPS14 Rabbit polyclonal,1:1000 Statens Anti CPS15b Rabbit polyclonal, 1:1000 Statens AntiPseudomonas O11 Rabbit polyclonal, 1:500 Lam lab, antigen University ofGuelph Anti Salmonella LT2 O Rabbit polyclonal, 1:1000 Statens antigenhR6 Rabbit polyclonal, 1:200000 Aebi lab Goat Anti rabbit IR680 1:15000Li-Cor Goat Anti mouse IR800 1:15000 Li-Cor HRP-conjugated IgM Mousemonoclonal 1:10000 Rockland HRP-conjugated IgG Mouse monoclonal 1:10000Rockland HRP-conjugated Rabbit polyclonal 1:5000 Rockland

Total Membrane Preparations

Cells grown overnight at 37° C. in Terrific broth were washed withphosphate buffered saline (PBS) buffer and resuspended in the samebuffer. Cells were lysed by two rounds of cell disruption using a Frenchpress at 20 kpsi followed by the addition of a protease inhibitorcocktail (Roche). Lysates were centrifuged for 30 minutes at 10000×g toget rid of cell debris and supernatants were then ultracentrifuged at100000×g for 60 minutes to pellet total membranes. The pellet wasresuspended in a buffer containing 0.1% Triton x100 or 1%n-dodecyl-β-D-maltoside (DDM) in 10 ml PBS and membrane proteins weresolubilized by tumbling overnight. An equal volume of PBS was added tothe suspension to reduce detergent concentration and the suspension wasultracentrifuged at 100000 G for 60 minutes. Supernatants, whichcorrespond to solubilized membrane proteins, were loaded on columns fornickel affinity protein purifications.

Nickel Affinity Protein Purifications

Hexa-histidine-tagged proteins were purified from solubilized totalmembranes by nickel affinity chromatography. Briefly, total membraneswere loaded on nickel-nitrilotriacetic acid (Ni-NTA) agarose columns(Qiagen) previously equilibrated with a buffer containing 20 mMimidazole. To remove unbound proteins, the column was washed four timeseach with buffers containing 20 mM and 30 mM imidazole. His-taggedproteins bound to the column were eluted over six fractions with anelution buffer containing 250 mM imidazole.

Alternatively, ÄKTA purifier (Amersham Biosciences, Sweden) was employedfor protein purifications. Solubilized membrane proteins were firstfiltered through 0.45 μm and 0.22 μm filters, and then loaded on aHis-Trap HP column (GE Healthcare) previously equilibrated with a buffercontaining 20 mM imidazole. Unbound proteins were removed by washing thecolumn ten times each with buffers containing 20 mM and 30 mM imidazole.To elute proteins bound to the column, gradient elutions with anincremental increase in imidazole concentration of the elution bufferwere used.

Mouse Immunizations:

Imidazole was removed by an overnight round of dialysis followed by 22-hour rounds in a dialysis buffer composed of 250 ml PBS containing0.25% DDM. Proteins were quantified using a DC kit (biorad) after whichthe samples were diluted to approximately 6 μg/ml and 0.6 μg injectedper mouse. Two groups of 10 mice were injected either unglycosylatedComP or CPS-conjugated ComP. Sera from the mice were obtained beforeimmunization, 7 and 21 days post immunization. A booster dose was givenon the 14^(th) day.

Whole Cell ELISAs.

S. pneumoniae serotype 14 (Statens Serum Institut, Denmark) was grownovernight in BHI broth at 37° C. with 5% CO2 aerobic conditions. Cellswere washed in 1×PBS and OD was adjusted to 0.6. Cells were then heatinactivated by incubation at 60° C. for 2 h followed by immobilizing onCorning high binding 96 well plates (50 plJwell). Plates were incubatedovernight at 4° C. The following day, wells were washed three times with1×PBS (100 pr/well) before blocking with 5% skimmed milk for 2 h. Thewells were washed three times with PBS. Plates were then incubated foran hour at room temperature with mouse sera (100 μL/well) at a 1:500dilution in 2.5% skimmed milk in PBST. For the positive control, acommercial rabbit polyclonal antibody against CPS of serotype 14 wasused (Statens serum institute). Negative control wells were treated withskimmed milk instead of the primary antibody. After incubation with theprimary antibody, wells were washed three times with PBS. This wasfollowed by a 1-hour incubation with secondary HRP-conjugated antibodies(100 μL/well). Antibodies employed were the anti mouse IgM (1:10000),anti mouse IgG (1:10000) and anti rabbit (1:5000) HRP-conjugatedantibodies diluted in 2.5% skimmed milk in PBST. After incubation, thewells were washed three times with PBS and 100 μL of the chromogenicsubstrate TMB (Cell Signaling Technology) was added to each well and theplate was incubated at room temperature for 5 minutes after which theabsorbance at 650 nm was measured using a BioTek™ plate reader.

Bacterial surface glycans such as capsular polysaccharides (CPS) and Oantigens are good vaccine candidates as they were demonstrated toprovide glycan-specific protection. Said glycans when used alone elicitT cell-independent immune responses, with no memory cells being formed,and subsequent booster doses are required to sustain protection. As aresult, the efficacy of glycan-only vaccines has been well documented inadults under 55 years. However, children <5 respond poorly, if at all,to these vaccines, which has been attributed to the low expression ofCD21 on the surface of B cells in the spleen and blood at this age. Inthe elderly, polysaccharide vaccines are less effective due to thephysiological age-associated atrophy of haematopoietic tissue andprimary lymphoid organs, causing a decreased production of B and T cells(Griffloen et al., 1991; Simell et al., 2008) (Reviewed by Pace, 2013).

Conjugate vaccines, where surface glycans are linked to immunogenicproteins, have greatly reduced incidence of diseases, compared toglycan-only vaccines. In conjugate vaccines, surface glycans areconjugated to immunogenic proteins, and this elicits T cell-dependentimmune responses, which are stronger immune responses that are alsoassociated with the development of memory cells for subsequentinfections. The efficacy of conjugate vaccines was demonstrated inchildren <5 years, which could be attributed to the fact that infant Tcells show adult immunophenotypes and mount equally robust immuneresponses to conjugate vaccine antigens (Timens et al., 1989). For thesereasons, conjugate vaccines are gaining momentum in the vaccine market(reviewed by Pace, 2013).

Most conjugate vaccines in the market today are synthesized bychemically conjugating glycans to proteins, which is an expensiveprocess with numerous drawbacks (Dick and Beurret, 1989; Peeters et al.,2003; Lees et al., 2006). Exploiting OTases to perform this conjugationin bacteria has been suggested as a method for cutting vaccinemanufacturing costs and improving the efficiency of the process (Terraet al., 2012). OTases heterologously expressed in a bacterial expressionsystem (usually engineered Escherichia coli strains) transferlipid-linked glycans to acceptor proteins, after which the glycoproteinis subsequently purified. Examples of OTase-conjugated vaccines include,but are not limited to, those against Pseudomonas aeruginosa,Francisella tularensis, Burkholderia pseudomallei, Shigella flexneri andShigella dysenteriae (Horzempa et al., 2008; Cuccui et al., 2013;Garcia-Quintanilla et al., 2014; Kimpf et al., 2015; Ravenscroft et al.,2015).

To date, the best-characterized OTases for producing conjugate vaccinesare the Campylobacter N-OTase PglB (CjPglB) and the O-OTases TfpO/PilOfrom Pseudomonas aeruginosa and NmPglL from Neisseria meningitidis. Thementioned OTases were found be specific towards the glycans transferredand the range of acceptor proteins they glycosylate.

Regarding acceptor proteins, CjPglB transfers glycans to Asn residues ofacceptor proteins that lie in the sequon D/EX₁NX₂S/T (SEQ ID NO: 110),with X being any amino acid except proline, and the residue in the −2position being acidic, namely glutamic acid (D) or aspartic acid (E)(Kowarik et al., 2006). A DQNAT sequon (SEQ ID NO: 111) was identifiedto be the optimal sequon for glycosylation by CjPglB (Chen et al.,2007). Insertion of this sequon, termed “glycotag”, at N or C termini ofunglycosylated proteins such as E. coli maltose-binding protein MalEinduced glycosylation by CjPglB (Fisher et al., 2010).

No consensus sequence has been identified for the glycosylation site ofO-OTases to date. Instead, the glycosylated residues identified were inlow complexity regions, with an abundance of serine, proline and alanineresidues (Vik et al., 2009). This means that acceptor proteins arelimited to the natural substrates of O-OTases, which in case of TfpO isthe P. aeruginosa strain 1244 Type IV pilin PilA and in case of NmPglLis a wide range of proteins. However, a similar idea to theN-glycosylation glycotags has been employed in P. aeruginosa, where TfpOwas able to glycosylate other proteins with the C terminal 15 residuesof PilA (where the glycosylation site lies) fused to their C terminal(Qutyan et al., 2010).

With regards to glycan specificity, CjPglB requires an acetamido groupat C-2 of the reducing end for glycan transfer to acceptor proteins.Furthermore, CjPglB was shown to only transfer glycans with N-acetylatedsugar residues at the reducing end of the glycan such as FucNAc, GalNAc,GlcNAc and Bac (Feldman et al., 2005; Wacker et al., 2006).Additionally, CjPglB was demonstrated to transfer Burkholderia.pseudomallei O polysaccharide II, the equivalent of O antigen. Thisglycan is a polymer of disaccharide repeating subunits composed ofglucose and O-acetyl deoxytalose (Garcia-Quintanilla et al., 2014).

TfpO/PilO was demonstrated to transfer multiple Pseudomonas 0 antigens,all of which are either tri or tetrasaccharides with FucNAc at thereducing end, to PilA. TfpO also transferred the E. coli 0157 antigen, atetrasaccharide with GalNAc at the reducing end (DiGiandomenico et al.,2002; Horzempa et al., 2006). Furthermore, TfpO was demonstrated to onlytransfer short chain oligosaccharides of the E. coli O7 antigen, apentasaccharide with GlcNAc at the reducing end, greatly limiting itspotential for the conjugate vaccine industry apart from Pseudomonasconjugate vaccines (Faridmoayer et al., 2007).

NmPglL on the other hand displayed a more relaxed glycan specificitythan TfpO, and was demonstrated to transfer sugars with N-acetylatedglycans at the reducing end as well as glycans with Gal residues at thereducing end, characteristic of the Salmonella enterica S. Typhimurium Oantigen (which could not be transferred by CjPglB). In addition tohaving different glycan and protein specificities, the O-OTases NmPglLand TfpO are sequentially and phylogenetically distinct, which suggeststhat they comprise two distinct classes of O-OTases. PglL-like OTasesappear to be far more superior and with more potential inglycoengineering than TfpO-like OTases, given their ability to transferlonger oligosaccharides and their more relaxed glycan specificity.

A limitation of all characterized OTases to date is that none of themare able to transfer glycans with glucose residues at the reducing end.Such glycans are characteristic of the capsular polysacharides ofmembers of the genus Streptococcus, an example of which being S.pneumoniae.

Streptococcus pneumoniae, or the Pneumococcus, is one of the leadingcauses of bacterial meningitis in infants and children. Children under5, the elderly and immunocompromised are at a particular high risk ofcommunity-acquired pneumococcal infections (Reviewed by Watson et al.,1993). In 2000, conservative estimates of Pneumococcus infectionspredicted 14.5 million cases. Pneumococcus-associated mortalities inchildren under 5 years are estimated to be 826000 annually, accountingfor approximately 11% of mortalities in children under 5 (O'Brien etal., 2009). More than 90 serotypes have been identified for thePneumococcus, each possessing a structurally and immunogenicallydistinct polysaccharide capsule that is the basis of pneumococcusvaccines (Lund, 1970; Kadioglu et al., 2008). Pneumococcus conjugatevaccines available in the market are produced by chemical conjugationsof CPS to the modified diphtheria toxin CRM₁₉7, which, despite itsexceptional efficacy, is significantly more expensive thanpolysaccharide-only vaccines according to the CDC vaccine price list.This has led the slow implementation of Pneumococcus vaccine programs bylow-income countries without external aid (Wenger, 2001; Weinberger etal., 2011).

As a result of this, an OTase capable of biologically conjugating theStreptococcus CPS to acceptor proteins, such as that of thePneumococcus, would revolutionize immunization against this organism,leading to significantly cheaper vaccines and ultimately leading to areduction in disease burden and child deaths especially in low-incomecountries.

In the present example there is provided an O-OTase that appears toconstitute a novel class of O-OTases. The OTase is called PglL_(ComP)and was identified in the non-pathogenic strain Acinetobacter baylyi(Harding et al., 2015). This OTase is phylogenetically and sequentiallysimilar to PglL-like OTases and transfers similar glycans (FIG. 12).However, it only has one acceptor protein, the type IV pilin ComP, afeature similar to the TfpO-like OTases. The present exampledemonstrates that PglL_(ComP) has the ability to transfer CPS from S.pneumoniae serotype 14—which has a glucose residue at the reducing end-to ComP, whereas all previously identified OTases are unable to transfersugars containing glucose at the reducing end (FIG. 13). This findingopens the door to a wide range of applications of PglL_(ComP) inmanufacturing Streptococcus conjugate vaccines, including but notlimited to S. pneumoniae but to other Streptococci, such as the swinepathogen S. suis and the Bovine pathogen S. uberis.

EXAMPLE 3

In the present example there is provided evidence that injection of CPS14-conjugated to ComP via PglL_(ComP) in E. coli mounts an IgG immuneresponse against S. pneumoniae serotype 14 capsule.

In this example, it was sought to determine whether ComP conjugated withthe S. pneumoniae serotype 14 capsular polysaccharide would generate animmunogenic response in a murine model. Overexpressed his-tagged ComPeither conjugated to CPS or in its unglycosylated form were purified byNickel affinity chromatography from E. coli SDB1 detergent-solubilizedtotal membranes. For immunization, 60 ng of CPS-conjugated ComP wereused per mouse (n=10) and as a control group, mice were immunized with60 ng of unglycosylated ComP each (n=10). A booster dose of theglycoprotein was administered on day 14 and pre immune sera and postimmune sera (day 7 and day 21) were collected. To determine if there wasan immunogenic response, we performed whole cell ELISAs by immobilizingheat-killed S. pneumoniae serotype 14 on 96 well plates. Pre immune andpost-immune sera from day 7 did not produce and immune response to thewhole cells. Post immune sera from day 21 of the mice injected withCPS-conjugated ComP demonstrated marginal IgM and significant IgG basedimmune responses against this serotype. An IgG immune response was notseen with the negative control mice injected with the glycosylated ComP(FIGS. 14, 15, 16A). No reactivity was seen in the negative controlwells (not treated with a primary antibody) whereas strong reactivitywas seen in wells treated with a commercially available anti CPS 14antibody (SSI Denmark) (FIG. 16B). Similar results were seen in Westernblots, where S. pneumoniae CPS expression from E. coli CLM37 was probedfor with mice sera (FIG. 17).

TABLE 6 Primers used in the present application Primer Set Sequence  1 FAGAATACTTGCATAGTGACAGGTTACAG (SEQ ID NO: 1) RGTTATGGCGGCGGTGGAGGTC (SEQ ID NO: 2)  2 FCAAAAAGCTTATATAAAAACATACATACAATCTTTGGGGAAAAGGCTATGATTCCGGGGATCCGTCGACC (SEQ ID NO: 3) RGGATTGACCTCTCTTTTTTATTTCTAAAATTACGATGCTACAAATGATTGTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 4)  3 FGCGGGATCCGCAAATTGGTGATGTGATGTCTCG (SEQ ID NO: 5) RGCGGGTACCGCTGCGAGGAATAAAAAGAATACT (SEQ ID NO: 6)  4 FGCGGGATCCGCAAATTGGTGATGTGATGTCTCG (SEQ ID NO: 7) RGCGGGTACCTCGTATTGTGAACTAGACCATCCT (SEQ ID NO: 8)  5 FGCGGGATCCGCAAATTGGTGATGTGATGTCTCG (SEQ ID NO: 9) RGCGGGTACCGCTGCGAGGAATAAAAAGAATACT (SEQ ID NO: 10)  6 FAGAATACTTGCATAGTGACAGGTTACAG (SEQ ID NO: 11) RCGCATTTATATTTGGGGATTACTC (SEQ ID NO: 12)  7 FCTTCCATGTATAATTCTTCTCAAGTTTTTGGTCTGTAACCTGTCACTATGATTCCGGGGATCCGTCGACC (SEQ ID NO: 13) RAAAATCCCCTTGAAAACAAGGGGATTTTTTTATTTATCTTTTAATAATTGTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 14)  8 FCTTCCTCAATCATTTGTAGCAGCGTAATTTTAGAAATAAAAAAG (SEQ ID NO: 15) RCTTTTTTATTTCTAAAATTACGCTGCTACAAATGATTGAGGAAG (SEQ ID NO: 16)  9 FATGAAAAAACTTGAGCACCTTGC (SEQ ID NO: 17) RTGTTTGCTCTTATTTCTACTG (SEQ ID NO: 18) 10 FTTGTCATTTATAAAGTTAGTCAC (SEQ ID NO: 19) RTGTACACCTGATTTTAATATTCTA (SEQ ID NO: 20) 11 FGAAATAAGAGCAAACAATTCCGGGGATCCGTCGACC (SEQ ID NO: 21) RCTTTATAAATGACAATGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 22) 12 FCTCAAGTTTTTTCATCGCCATGGCGGCCGGGAGCATG (SEQ ID NO: 23) RAAAATCAGGTGTACAACTAGTGAATTCGCGGCCGCCTGCA (SEQ ID NO: 24) 13 FCGTCCCCAAAAGCGTGAA (SEQ ID NO: 25) RTTAGGCAAATTTCGAAGCGTGAT (SEQ ID NO: 26) 14 FGCGCCCGGGATAAGTGCTCAATTGATGG (SEQ ID NO: 27) RGGTACCGAGATCCCAAACCAGCAAC (SEQ ID NO: 28) 15 FACTAGTGAATTCGCGGCCGCCTGCA (SEQ ID NO: 29) RCGCCATGGCGGCCGGGAGCATG (SEQ ID NO: 30) 16 FATTCCGGGGATCCGTCGACC (SEQ ID NO: 31) RTGTAGGCTGGAGCTGCTTCG (SEQ ID NO: 32) 17 FCCGGCCGCCATGGCGATGACGATTGGTTTAATTTTTTC (SEQ ID NO: 33) RACGGATCCCCGGAATCATACTTGTAAAAAAAAAAGTATTT (SEQ ID NO: 34) 18 FCAGCTCCAGCCTACAATGGAAGAAAATTCTTTATTAATTT (SEQ ID NO: 35) RCGCGAATTCACTAGTTTAAACATATTTTTCCCATTTC (SEQ ID NO: 36) 19 FCCGGCCGCCATGGCGATGACTCCTGCCGGAGG (SEQ ID NO: 37) RACGGATCCCCGGAATTTAATAAAGAATTTTCTTCCATTTAC (SEQ ID NO: 38) 20 FCAGCTCCAGCCTACAATAGTAGGACTAAAAAAATGATTTCG (SEQ ID NO: 39) 20 RCGCGAATTCACTAGTTTATTTATATAACCCTTTTTCTTTC (SEQ ID NO: 40) 21 FCGGCCGCCATGGCGATGTTTAAAAATGTATTAATTACTGG (SEQ ID NO: 41) RACGGATCCCCGGAATCATTTATTTATATAACCCTTTTTCT (SEQ ID NO: 42) 22 FCAGCTCCAGCCTACAATAAATTTAAAATATTCATAAATCT (SEQ ID NO: 43) RCGCGAATTCACTAGTTTATAATTTAAGTTCTTGAATCAAC (SEQ ID NO: 44) 23 FCTACATTGTTTATTTTTACCAGAA (SEQ ID NO: 45) RGAAGCTTGAAGTTATCCACGAA (SEQ ID NO: 46) 24 FCATCAAAAATACCAGCCTAAATTATC (SEQ ID NO: 47) RCCATTGTTTGAAATTATTTAGGG (SEQ ID NO: 48) 25 FCATCAAAAATACCAGCCTAAATTATC (SEQ ID NO: 49) RGAAGAAAATTCTTTATTAATTTCTG (SEQ ID NO: 50) 26 FCATCAAAAATACCAGCCTAAATTATC (SEQ ID NO: 51) RGAAAAAGGGTTATATAAATAAATG (SEQ ID NO: 52) 27 FGCGCCCGGGCTACATTGTTTATTTTTACCAGAA (SEQ ID NO: 53) RGCGGGTACCACCATCATTGACTACTAAGACCTC (SEQ ID NO: 54) 28 FGCGCCCGGGCTACATTGTTTATTTTTACCAGAA (SEQ ID NO: 55) RGCGGGTACCTTCTACATCCAATACCAGTCGT (SEQ ID NO: 56) 29 FGCGCCCGGGCTACATTGTTTATTTTTACCAGAA (SEQ ID NO: 57) RGCGGGTACCGAAGCTTGAAGTTATCCACGAA (SEQ ID NO: 58) 30 FGCGCCCGGGCCGAAGCAGGGTGGGTGTTAGT (SEQ ID NO: 59) RGCGGGTACCTTAGTGGTGGTGGTGGTGTTGAGCTACTGAAAACTCAATAC (SEQ ID NO: 60) 31 FGCCATATGGCTTATCAAAACTATATTGCTAAATCTC (SEQ ID NO: 61) RGCGGATCCCTCTTTTTTATTTCTAAAATTACGATGCT (SEQ ID NO: 62) 32 FATAGGATCCATGAATGCACAAAAGGGTTTTACC (SEQ ID NO: 63) RTATGTCGACTCAGTGGTGGTGGTGGTGGTGACCACGACATTCTGATGG (SEQ ID NO: 64) 33 FATATGGATCCGTGGTTGATAGTAGTACTATATGG (SEQ ID NO: 65) RATATGTCGACTCAGTGGTGGTGGTGGTGGTGATATTCTATTGAACAAAATTTTAACTTAGG (SEQ ID NO: 66) 34 FATATGGATCCGTGGCTGGTTCCCCGCGTGTGTATAATAGC (SEQ ID NO: 67) RATATGTCGACTTAGTGGTGGTGGTGGTGGTGCTTGGATGACTCTACAGCAGAAGC (SEQ ID NO: 68)35 F ACTGGGATCCATGTTAAAAAAAATTATTCTATTTC (SEQ ID NO: 69) RACTGGTCGACTAAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGAGGAATTTTTGAGGTTGGTAC (SEQ ID NO: 70) 36 FACTGGGATCCATGCAAGTATTCTTTCTGTTC (SEQ ID NO: 71) RACTGGTCGACTAAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGTTTATTTTTTAACAACTCTGCC (SEQ ID NO: 72) 37 F GGTGGACGTGGAGGAG (SEQ ID NO: 73) RCTTGCTTGGGTTACATCAGTGCT (SEQ ID NO: 74) 38 FAATTATTGTACAGCCTTTTG (SEQ ID NO: 75) RCATCATCATCATCATCACTAATATTAAAAATGTATAAAAAACACC (SEQ ID NO: 76) 39 FCCAGTTGAATTACTTCCTCAAGCATTTGTAGCATCGTAAT (SEQ ID NO: 77) RATTACGATGCTACAAATGCTTGAGGAAGTAATTCAACTGG (SEQ ID NO: 78)

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All publications, patents and patent applications mentioned in thisSpecification are indicative of the level of skill of those skilled inthe art to which this invention pertains and are herein incorporated byreference to the same extent as if each individual publication, patent,or patent applications was specifically and individually indicated to beincorporated by reference.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method ofglycosylating a protein in a bacterial cell, the method comprisingglycosylating a fusion protein comprising ComP, with a bacterialcapsular polysaccharide containing glucose at its reducing end, using aPglL_(comP) O-oligosaccharyltransferase.
 2. The method of claim 1comprising expressing PglL_(comP) O-oligosccharyltransferase, the ComPfusion protein, and the capsular polysaccharide together in thebacterial cell.
 3. The method of claim 1, wherein the ComP fusionprotein comprises an adjuvant or carrier.
 4. The method of claim 1,wherein the bacterial cell is Acinetobacter or E. coli.
 5. The method ofclaim 1, wherein the capsular polysaccharide is a Streptococcus capsule.6. The method of claim 1, wherein the capsular polysaccharide is aStreptococcus pneumoniae capsule.
 7. The method of claim 4, wherein theAcinetobacter is A. baylyi, A. baumannii, A. nosocomialis, or A.calcoaceticus.
 8. A conjugate vaccine comprising the glycosylated ComPfusion protein produced in accordance with the method of claim
 1. 9. Theconjugate vaccine of claim 8, wherein the capsular polysaccharide is aStreptococcus capsule and the vaccine is a Streptococcus vaccine.
 10. Aglycosylated fusion protein comprising ComP, wherein the fusion proteinis glycosylated with a bacterial capsular polysaccharide containingglucose at its reducing end.
 11. The method of claim 4, wherein thebacterial cell is E. coli.
 12. The conjugate vaccine of claim 8, whereinthe capsular polysaccharide is a Streptococcus pneumoniae capsule andthe vaccine is a Streptococcus pneumoniae vaccine.
 13. The conjugatevaccine of claim 8, wherein the vaccine comprises an adjuvant orcarrier.
 14. The glycosylated fusion protein of claim 10, wherein thecapsular polysaccharide is from Streptococcus.
 15. The glycosylatedfusion protein of claim 10, wherein the capsular polysaccharide is fromStreptococcus pneumoniae.
 16. A conjugate vaccine comprising theglycosylated ComP fusion protein of claim
 10. 17. The conjugate vaccineof claim 16, wherein the capsular polysaccharide is a Streptococcuscapsule and the vaccine is a Streptococcus vaccine.
 18. The conjugatevaccine of claim 16, wherein the capsular polysaccharide is aStreptococcus pneumoniae capsule and the vaccine is a Streptococcuspneumoniae vaccine.
 19. The conjugate vaccine of claim 16, wherein thevaccine comprises an adjuvant or carrier.